Derivatized hyperbranched polyglycerols

ABSTRACT

Herein are provided derivatized hyperbranched polyglycerols (“dHPGs”). The dHPG comprises a core comprising a hyperbranched polyglycerol derivatized with C1C20 alkyl chains and a shell comprising at least one hydrophilic substituent bound to hydroxyl groups of the core, wherein the hyperbranched polyglycerol comprises from about 1 to about 200 moles of the at least one hydrophilic substituent. The dHPGs are for use as agents for the delivery of a drug or other biologically active moiety to the urinary tract, the digestive tract, the airways, the vaginal cavity and cervix and the peritoneal cavity to treat indications such as cancer, which may be useful in the treatment of or the manufacture of a medicament, in the preparation, of a pharmaceutical composition for the treatment of cancer, as a pre-treatment or co-treatment to improve drug uptake in a tissue. Furthermore, there are provided methods of making dHPGs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/868,148, filed Sep. 28, 2015, now U.S. Pat. No. 9,561,278, which is acontinuation of U.S. application Ser. No. 13/581,463, filed Jan. 18,2013, now U.S. Pat. No. 9,186,410, which is a National Stage entry ofInternational Application No. PCT/CA2011/000225, filed Mar. 1, 2011,which claims the benefit of U.S. Provisional Patent Application Ser. No.61/309,304 entitled “BIOADHESIVE DERIVATIZED HYPERBRANCHEDPOLYGLYCEROLS” filed on Mar. 1, 2010, the disclosures of each of whichare incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention relates to therapeutics, their uses and methods for thedelivery of drugs or other biologically active moieties to biologicaltissues. In particular, the invention relates to polymers based onderivatized hyperbranched polyglycerols (dHPGs) and methods for treatingcancer, infections and inflammatory or autoimmune diseases.

BACKGROUND

Bladder cancer is the second most common genitourinary malignancy. Atinitial diagnosis, approximately 70% of cases are non-muscle-invasive.Current treatment options for superficial disease include treatingbladder cancer topically via intravesicular instillation of achemotherapeutic agent into the bladder with a catheter. However, thesetreatment options are of limited efficacy. Despite intravesicalchemotherapy and/or immunotherapy, up to 80% of patients withnon-muscle-invasive bladder cancer develop recurrent tumours, of which20-30% develop into more aggressive, potentially lethal tumours(Dalbagni, G. (2007) Nat. Clin. Pract. Urol. 4: 254-260).

Treatment failure is thought to be due in part to the short dwell-timeof drugs active against bladder cancer cells in the bladder. Forexample, taxanes are generally not used for intravesicular instillationdue to poor bioavailability of the current formulations in the bladder.Paclitaxel has documented antitumor activity in systemic bladder cancertherapy as it penetrates bladder tissues at a rate 20 times faster thanthat of water-soluble drugs such as mitomycin C, allowing for prolongedretention of therapeutic doses even after the instilled solution isremoved. However, its intravesical use is hampered by the presence ofCremophor™-EL in the commercial formulation (Taxol™) as it entraps thedrug in an aqueous environment and reduces paclitaxel penetration intothe bladder wall (Mugabe, C., et al. (2008) British J. Urology Int.102(7): 978-986).

While many active agents are hydrophobic or otherwise water insoluble,they are often needed in water-based or otherwise aqueous environmentsfor effective treatment of numerous indications, including cancer (suchas cancers of the bowel, lung, bladder and genitourinary system),infections (such as those of the digestive tract and the airways) andinflammatory or autoimmune diseases (such as irritable bladder,inflammatory bowel disease and chronic and acute inflammation). As such,multiple systems have been developed as delivery vehicles for suchagents. One of these systems includes the use of polymeric micelles.

Polymeric micelles are amphiphilic, having a hydrophobic core and ahydrophilic shell, and as such, they can encapsulate hydrophobicmolecules in the core due to hydrophobic interactions. The hydrophilicshell keeps the system soluble in water. However, these systems may beunstable in the bladder due to dilution effects or environmentalfactors.

Hyperbranched polyglycerols (“HPGs”) are one of the few hyperbranchedpolymers that can be synthesized in a controlled manner withpre-determined molecular weights and narrow polydispersity (Kainthan, R.K., et al. (2008) Biomacromolecules 9: 886-895). Hydrophobic moleculesmay be encapsulated in the hydrophobic core of an HPG (WO2006/130978).

SUMMARY

This invention is based in part on the discovery that derivatizedhyperbranched polyglycerols (“dHPGs”) described herein may be used asagents for the delivery of a drug or other biologically active moiety tothe urinary tract (for example, the urethra and bladder), the digestivetract (for example, the mouth, esophagus and colon), the airways (forexample, the nose and lungs), the vaginal cavity and cervix and theperitoneal cavity to treat indications such as cancer (for example,bladder, gastric, esophageal, lung, laryngeal, oral, sinus, vaginal orcervical cancers), infection (for example, infections of the digestivetract or the airways), and inflammatory or autoimmune diseases (forexample, irritable bladder, inflammatory bowel disease or chronic oracute inflammation) as well as other indications wherein delivery of adrug or other biologically active moiety to a tissue or cell is desired.For example, polymers identified herein may be useful in instillationtherapy of non-muscle-invasive bladder cancer. Polymers identifiedherein may show mucoadhesive properties which may be useful ininstillation therapy of non-muscle-invasive bladder cancer.

The dHPGs herein described may be used as a carrier for a drug or otherbiologically active moiety and for the preparation of a therapeuticmedicament for delivery of such drugs or moieties to target tissues orcells. In particular, the dHPGs herein described may be used as acarrier for a taxane for the treatment of non-muscle-invasive bladdercancer.

The condensed core dHPGs described herein have surprising attributesthat are particularly desirable for delivery of a drug to a targettissue. In particular, as shown herein, condensed core dHPGs are lesstoxic and have greater tolerability properties.

In accordance with an embodiment, there is provided a hyperbranchedpolyglycerol, the hyperbranched polyglycerol comprising: a corecomprising hyperbranched polyglycerol derivatized with C₁-C₂₀ alkylchains, wherein the ratio of C₁-C₂₀ alkyl chains to glycerol units isgreater at a centre of the core compared to a periphery of the core; anda shell comprising at least one hydrophilic substituent bound tohydroxyl groups of the core, wherein the hyperbranched polyglycerolcomprises from about 1 to about 200 moles of the at least onehydrophilic substituent per mole of the hyperbranched polyglycerol.

In accordance with another embodiment, there is provided a method ofdelivering a biologically active moiety to a biological tissue, themethod comprising: administering a hyperbranched polyglycerol loadedwith the biologically active moiety to the biological tissue, whereinthe hyperbranched polyglycerol comprises: a core comprisinghyperbranched polyglycerol derivatized with C₁-C₂₀ alkyl chains, whereinthe ratio of C₁-C₂₀ alkyl chains to glycerol units is greater at acentre of the core compared to a periphery of the core; and a shellcomprising at least one hydrophilic substituent bound to hydroxyl groupsof the core, wherein the hyperbranched polyglycerol comprises from about1 to about 200 moles of the at least one hydrophilic substituent permole of the hyperbranched polyglycerol. The method may further compriseincorporating the biologically active moiety into the hyperbranchedpolyglycerol.

In accordance with a further embodiment, there is provided a use of ahyperbranched polyglycerol for delivering a biologically active moietyto a biological tissue, wherein the hyperbranched polyglycerolcomprises: a core comprising hyperbranched polyglycerol derivatized withC₁-C₂₀ alkyl chains, wherein the ratio of C₁-C₂₀ alkyl chains toglycerol units is greater at a centre of the core compared to aperiphery of the core; and a shell comprising at least one hydrophilicsubstituent bound to hydroxyl groups of the core, wherein thehyperbranched polyglycerol comprises from about 1 to about 200 moles ofthe at least one functional group per mole of the hyperbranchedpolyglycerol.

In accordance with another embodiment, there is provided a use of ahyperbranched polyglycerol for preparing a medicament for delivering abiologically active moiety to a biological tissue, wherein thehyperbranched polyglycerol comprises: a core comprising hyperbranchedpolyglycerol derivatized with C₁-C₂₀ alkyl chains, wherein the ratio ofC₁-C₂₀ alkyl chains to glycerol units is greater at a centre of the corecompared to a periphery of the core; and a shell comprising at least onehydrophilic substituent bound to hydroxyl groups of the core, whereinthe hyperbranched polyglycerol comprises from about 1 to about 200 molesof the at least one functional group per mole of the hyperbranchedpolyglycerol.

In accordance with a further embodiment, there is provided apharmaceutical composition comprising a hyperbranched polyglycerol and abiologically active moiety, wherein the hyperbranched polyglycerolcomprises: a core comprising hyperbranched polyglycerol derivatized withC₁-C₂₀ alkyl chains, wherein the ratio of C₁-C₂₀ alkyl chains toglycerol units is greater at a centre of the core compared to aperiphery of the core; and a shell comprising at least one hydrophilicsubstituent bound to hydroxyl groups of the core, wherein thehyperbranched polyglycerol comprises from about 1 to about 200 moles ofthe at least one functional group per mole of the hyperbranchedpolyglycerol.

In accordance with an embodiment, there is provided a hyperbranchedpolyglycerol, comprising: a core comprising hyperbranched polyglycerolderivatized with C₁-C₂₀ alkyl chains; and a shell comprising at leastone hydrophilic substituent bound to hydroxyl groups of the core,wherein the at least one hydrophilic substituent comprises at least onefunctional group selected from one or more of the following: —NH₂, ═NH₂⁺, —NH₃ ⁺, and —NR₃ ⁺, wherein each R is independently a C₁-C₆ alkylgroup or one R is independently a C₁-C₆ alkyl group and two R's togetherform a C₃-C₁₂ cyclic alkyl group so that R₃ forms a quaternary aminewith the nitrogen, and wherein the hyperbranched polyglycerol comprisesfrom about 1 to about 200 moles of the at least one functional group permole of the hyperbranched polyglycerol.

In accordance with a further embodiment, there is provided a use of ahyperbranched polyglycerol, the hyperbranched polyglycerol comprising: acore comprising hyperbranched polyglycerol; and a shell comprising atleast one hydrophilic substituent bound to hydroxyl groups of the core,wherein the at least one hydrophilic substituent comprises at least onefunctional group selected from one or more of the following: —NH₂, ═NH₂⁺, —NH₃ ⁺, and —NR₃ ⁺, wherein each R is independently a C₁-C₆ alkylgroup or one R is independently a C₁-C₆ alkyl group and two R's togetherform a C₃-C₁₂ cyclic alkyl group so that R₃ forms a quaternary aminewith the nitrogen, and wherein the hyperbranched polyglycerol comprisesfrom about 1 to about 200 moles of the at least one functional group permole of the hyperbranched polyglycerol, for use as a pre-treatment orco-treatment to increase drug uptake in a tissue. In an embodiment, thecore may be further derivatized with C₁-C₂₀ alkyl chains.

In accordance with another embodiment, there is provided a hyperbranchedpolyglycerol, comprising: a core comprising hyperbranched polyglycerol;and a shell comprising at least one hydrophilic substituent bound tohydroxyl groups of the core, wherein the at least one hydrophilicsubstituent comprises at least one functional group selected from one ormore of the following: —NH₂, ═NH₂ ⁺, —NH₃ ⁺, and —NR₃ ⁺, wherein each Ris independently a C₁-C₆ alkyl group or one R is independently a C₁-C₆alkyl group and two R's together form a C₃-C₁₂ cyclic alkyl group sothat R₃ forms a quaternary amine with the nitrogen, and wherein thehyperbranched polyglycerol comprises from about 1 to about 200 moles ofthe at least one functional group per mole of the hyperbranchedpolyglycerol, for use as a pre-treatment or co-treatment to increasedrug uptake in a tissue. The core may be further derivatized with C₁-C₂₀alkyl chains. In an embodiment, increasing drug uptake in a tissue maycause loss of umbrella cells of the tissue. In an embodiment, increasingdrug uptake in a tissue may be without causing necrosis and/orinflammation of the tissue.

In accordance with another embodiment, there is provided a hyperbranchedpolyglycerol, the hyperbranched polyglycerol comprising: a corecomprising hyperbranched polyglycerol polymerized from a glycerolepoxide and a C₁-C₂₀ alkyl epoxide or a C₁-C₂₀ alkyl glycidyl ether,wherein all or substantially all of the C₁-C₂₀ alkyl epoxide or C₁-C₂₀alkyl glycidyl ether is polymerized before all or substantially all ofthe glycerol epoxide is polymerized; and a shell comprising at least onehydrophilic substituent covalently bonded to hydroxyl groups of thecore, wherein the hyperbranched polyglycerol comprises from about 1 toabout 200 moles of the at least one functional group per mole of thehyperbranched polyglycerol. In accordance with a further embodiment,there is provided a method of synthesizing a hyperbranched polyglycerol,the method comprising: polymerizing a glycerol epoxide and a C₁-C₂₀alkyl epoxide or a C₁-C₂₀ alkyl glycidyl ether such that all orsubstantially all of the C₁-C₂₀ alkyl epoxide or C₁-C₂₀ alkyl glycidylether is polymerized before all or substantially all of the glycerolepoxide is polymerized to form hyperbranched polyglycerol; andderivatizing hydroxyl groups of the hyperbranched polyglycerol with atleast one hydrophilic substituent, wherein the hyperbranchedpolyglycerol comprises from about 1 to about 200 moles of the at leastone functional group per mole of the hyperbranched polyglycerol. Theglycerol epoxide may be glycidol. The C₁-C₂₀ alkyl epoxide may be1,2-epoxyoctadecane. The C₁-C₂₀ alkyl glycidyl ether may be C₈-C₁₀ alkylglycidyl ether.

In accordance with another embodiment, there is provided a hyperbranchedpolyglycerol, the hyperbranched polyglycerol comprising: a corecomprising hyperbranched polyglycerol derivatized with C₁-C₂₀ alkylchains and loaded with docetaxel; and a shell comprising at least onehydrophilic substituent bound to hydroxyl groups of the core, whereinthe hyperbranched polyglycerol comprises from about 1 to about 200 molesof the at least one functional group per mole of the hyperbranchedpolyglycerol.

In accordance with a further embodiment, there is provided a use of ahyperbranched polyglycerol for delivering docetaxel to a biologicaltissue, wherein the hyperbranched polyglycerol comprises: a corecomprising hyperbranched polyglycerol derivatized with C₁-C₂₀ alkylchains and loaded with docetaxel; and a shell comprising at least onehydrophilic substituent bound to hydroxyl groups of the core, whereinthe hyperbranched polyglycerol comprises from about 1 to about 200 molesof the at least one functional group per mole of the hyperbranchedpolyglycerol.

The hyperbranched polyglycerol may further include a biologically activemoiety. The hyperbranched polyglycerol may be used as a pretreatment orco-treatment for increasing drug uptake of a biologically active moiety.The biologically active moiety may be one or more hydrophobic drugs. Thebiologically active moiety may be selected from one of more ofvalrubicin, cisplatin, paclitaxel, docetaxel. The biologically activemoiety may be a taxane or an analog thereof. The taxane may bepaclitaxel or an analog thereof. The taxane may be docetaxel or ananalog thereof. The biologically active moiety may be mitomycin or ananalog thereof. Mitomycin may include all mitomycin analogs. Mitomycinand analogs thereof may include, for example, mitomycin A, mitomycin B,mitomycin C, mitomycin D, mitomycin F, mitomycin G, mitomycin H,mitomycin K and analogs thereof. The biologically active moiety may bemitomycin C. The biologically active moiety may be mitomycin F. Thebiologically active moiety may be valrubicin. The biologically activemoiety may be vinblastine. The biologically active moiety may becisplatin. The biologically active moiety may be methotrexate. Thebiologically active moiety may be doxorubicin or an analog thereof. Thebiologically active moiety may be epirubicin. The biologically activemoiety may be gemcitabine. The biologically active moiety may beeverolimus. The biologically active moiety may be suramin. Thebiologically active moiety may be a combination of moieties. Thecombination of moieties may be methotrexate, vinblastine, anddoxorubicin (M-VAC). The combination of moieties may be M-VAC andcisplatin.

The hydrophilic substituent may be polyethylene glycol (PEG) (200 to 450g/ml), or methoxy polyethylene glycol (MPEG) (200 to 450 g/ml), orcombinations thereof. The at least one hydrophilic substituent may beMePEG or PEG. The at least one hydrophilic substituent may be MePEG. Theat least one hydrophilic substituent may be PEG. The at least onehydrophilic substituent may comprise at least one functional group thatis —OH, —COOH, —NHS, —SH, —NH₂, —NH₃ ⁺, or —NR₃+, wherein each R mayindependently be a C₁-C₆ alkyl group or one R may independently be aC₁-C₆ alkyl group and two R's together may form a C₃-C₁₂ cyclic alkylgroup so that R₃ forms a quaternary amine with the nitrogen.

The at least one functional group may be —NH₂, —NH₃ ⁺, or —NR₃+, whereineach R may independently be a C₁-C₆ alkyl group or one R mayindependently be a C₁-C₆ alkyl group and two R's together may form aC₃-C₁₂ cyclic alkyl group so that R₃ forms a quaternary amine with thenitrogen. The at least one functional group may be —NH₂, or —NH₃ ⁺. Theat least one functional group may be an amine. Alternatively, the atleast one functional group may be —NH₂.

The hyperbranched polyglycerol may comprise from about 1 to about 200moles of the at least one hydrophilic substituent per mole of thehyperbranched polyglycerol. The hyperbranched polyglycerol may comprisefrom about 1 to about 100 moles of the at least one hydrophilicsubstituent per mole of the hyperbranched polyglycerol. Thehyperbranched polyglycerol may comprise from about 1 to about 40 molesof the at least one hydrophilic substituent per mole of thehyperbranched polyglycerol. The hyperbranched polyglycerol may comprisefrom about 5 to about 40 moles of the at least one hydrophilicsubstituent per mole of the hyperbranched polyglycerol. Thehyperbranched polyglycerol may comprise from about 10 to about 40 molesof the at least one hydrophilic substituent per mole of thehyperbranched polyglycerol. The hyperbranched polyglycerol may comprisefrom about 10 to about 30 moles of the at least one hydrophilicsubstituent per mole of the hyperbranched polyglycerol. Thehyperbranched polyglycerol may comprise from about 30 to about 40 molesof the at least one hydrophilic substituent per mole of thehyperbranched polyglycerol. The hyperbranched polyglycerol may comprisefrom about 5 to about 15 moles of the at least one hydrophilicsubstituent per mole of the hyperbranched polyglycerol. The at least onehydrophilic substituent may bind to about 1% to about 40% of thehydroxyl groups. The at least one hydrophilic substituent may bind toabout 5% to about 30% of the hydroxyl groups. The at least onehydrophilic substituent may bind to about 20% of the hydroxyl groups.

The amount of the hydrophilic substituent per mol of the hyperbranchedpolyglycerol may be determined by measuring the molecular weight of thehyperbranched polyglycerol and measuring the amount of the hydrophilicsubstituent present in an amount of the hyperbranched polyglycerol. Theperson of ordinary skill in the art will appreciate that the molecularweight of the hyperbranched polyglycerol may be measured using differentmethods, for example, gel permeation chromatography. The molecularweight of the hyperbranched polyglycerol may be measured, for example,using gel permeation chromatography with multi-angle laser lightscattering detection. The amount of the hydrophilic substituent presentin an amount of the hyperbranched polyglycerol may be measured, forexample, by a titration method. The titration method may be a forwardtitration method. The titration method may be a back titration method.For example, where the hydrophilic substituent comprises at least onefunctional group that may be —NH₂, a forward titration method against anacid, such as HCl, may be used to measure the amount of hydrophilicsubstituent present in an amount of HPG. Where the hydrophilicsubstituent comprises at least one functional group that may be —NH₂, aback titration method using a known amount of an acid, such as HCl, andtitrating against a base, such as NaOH, may be used. The amount of thehydrophilic substituent present in an amount of hyperbranchedpolyglycerol may be measured, for example, by a colorimetric method. Theamount of the hydrophilic substituent present in an amount ofhyperbranched polyglycerol may be measured, for example, by afluorescence method. Where the hydrophilic substituent comprises atleast one functional group that may be —NH₂, a fluorescamine assay maybe used. The amount of the hydrophilic substituent present in an amountof hyperbranched polyglycerol may be measured by more than one methodand an average value of the amount of the hydrophilic substituentmeasured by the more than one methods may be used to calculate the molof hydrophilic substituent per mol of hyperbranched polyglycerol. Wherethe hydrophilic substituent comprises at least one functional group thatmay be —NH₂, a fluorescamine assay may be the preferred method todetermine the amount of the hydrophilic substituent present in an amountof hyperbranched polyglycerol.

The hyperbranched polyglycerol may comprise from about 1 to about 200moles of the at least one functional group per mole of the hyperbranchedpolyglycerol. The hyperbranched polyglycerol may comprise from about 1to about 100 moles of the at least one functional group per mole of thehyperbranched polyglycerol. The hyperbranched polyglycerol may comprisefrom about 1 to about 40 moles of the at least one functional group permole of the hyperbranched polyglycerol. The hyperbranched polyglycerolmay comprise from about 5 to about 40 moles of the at least onefunctional group per mole of the hyperbranched polyglycerol. Thehyperbranched polyglycerol may comprise from about 10 to about 40 molesof the at least one functional group per mole of the hyperbranchedpolyglycerol. The hyperbranched polyglycerol may comprise from about 10to about 30 moles of the at least one functional group per mole of thehyperbranched polyglycerol. The hyperbranched polyglycerol may comprisefrom about 30 to about 40 moles of the at least one functional group permole of the hyperbranched polyglycerol. The hyperbranched polyglycerolmay comprise from about 5 to about 15 moles of the at least onefunctional group per mole of the hyperbranched polyglycerol.

The amount of the functional group per mol of the hyperbranchedpolyglycerol may be determined by measuring the molecular weight of thehyperbranched polyglycerol and measuring the amount of the functionalgroup present in an amount of the hyperbranched polyglycerol. The personof ordinary skill in the art will appreciate that the molecular weightof the hyperbranched polyglycerol may be measured using differentmethods, for example, gel permeation chromatography. The molecularweight of the hyperbranched polyglycerol may be measured, for example,using gel permeation chromatography with multi-angle laser lightscattering detection. The amount of the functional group present in anamount of the hyperbranched polyglycerol may be measured, for example,by a titration method. The titration method may be a forward titrationmethod. The titration method may be a back titration method. Forexample, where the at least one functional group may be —NH₂, a forwardtitration method against an acid, such as HCl, may be used to measurethe amount of —NH₂ per present in an amount of HPG. Where the at leastone functional group may be —NH₂, a back titration method using a knownamount of an acid, such as HCl, and titrating against a base, such asNaOH, may be used. The amount of the functional group present in anamount of hyperbranched polyglycerol may be measured, for example, by acolorimetric method. The amount of the functional group present in anamount of hyperbranched polyglycerol may be measured, for example, by afluorescence method. Where the at least one functional group may be—NH₂, a fluorescamine assay may be used. The amount of the functionalgroup present in an amount of hyperbranched polyglycerol may be measuredby more than one method and an average value of the amount of thefunctional group measured by the more than one methods may be used tocalculate the mol of functional group per mol of hyperbranchedpolyglycerol. Where the at least one functional group may be —NH₂, afluorescamine assay may be the preferred method to determine the amountof the functional group present in an amount of hyperbranchedpolyglycerol.

The C₁-C₂₀ alkyl chains may be C₅-C₂₀ alkyl chains. The C₁-C₂₀ alkylchains may be C₈-C₁₈ alkyl chains. The C₁-C₂₀ alkyl chains may be C₈-C₁₀alkyl chains.

A portion of the at least one hydrophilic substituent may be located inthe core.

The biological tissue may be a mucosal membrane. The biological tissuemay be a cell. The biological tissue may be the urothelial surface of abladder.

The dHPGs as described herein may be described through a commonnomenclature which identifies the basic hyperbranched structure, thecore attributes, and the surface attributes as follows:HPG-core(x)-shell₁(y1)-shell₂(y2) . . . -shell_(n)(yn)  (I)

which designates a polymer composed of hyperbranched polyglycerol,comprising a core derivatized a substituent selected from hydrophobicgroups such as C₈, C₁₀, C₁₂ or C₁₈ alkyl groups that are either linearor branched or contain aryl substituents, wherein the amount of the coresubstituent is x, expressed in number of moles or as a percentage. Thepolymer also has n substituents on the shell, such as PEG or MePEG, orsubstituents having carboxyl groups (COOH), hydroxyl groups, amines(NR₂), N-hydroxysuccinimides (NHS), charged amines (NR₃ ⁺), thiols (SH)etc., as described herein. Each shell substituent may be designated asbeing present in a certain amount y1, y2 or yn and can be expressed innumber of moles or as a percentage. In some notations, general classescan be designated in the same manner, but without explicitly identifyingthe amounts of each. In addition, when the shell substituent is PEG orMePEG, it may be further defined by the chain length of this polymericcomponent, for example MePEG350, PEG200, etc. For the general classhowever, the molecular weight may be omitted.

For example, HPG-C_(8/10)-MePEG or HPG-C_(8/10)-NH₂ each designate thecore (x) as C_(8/10). The term “HPG-C_(8/10)-MePEG”, or the like,anywhere herein may be used interchangeably with the term“HPG-C10-MePEG”. In some circumstances the core(x) is not identified andmay be assumed to be C_(8/10). Nevertheless, other alkyls having C₁-C₂₀may be used. In accordance with a further embodiment, there is provideda use of a dHPG described herein for drug delivery to a target tissue.In accordance with a further embodiment, there is provided a use of adHPG described herein in the preparation of a medicament for drugdelivery to a target tissue. In accordance with a further embodiment,there is provided a use of a dHPG described herein as a pre-treatment orco-treatment for increasing drug uptake in a tissue. In accordance witha further embodiment, there is provided a use of a dHPG described hereinfor the treatment of non-muscle-invasive bladder cancer. In accordancewith a further embodiment, there is provided a use of a dHPG describedherein as a pre-treatment or co-treatment for increasing drug uptake ina tissue of a drug for the treatment of non-muscle-invasive bladdercancer. In accordance with a further embodiment, there is provided a useof a dHPG described herein in the preparation of a medicament for thetreatment of non-muscle-invasive bladder cancer. The treatment of thenon-muscle-invasive bladder cancer may be in a mammal. The mammal may behuman. In accordance with another embodiment, there is provided apharmaceutical composition comprising a dHPG as set out herein and apharmaceutically acceptable excipient. In accordance with a furtherembodiment, there is provided one or more of the dHPGs described hereinfor drug delivery to a target tissue. In accordance with a furtherembodiment, there is provided a method for preparing a dHPG describedherein.

The polymers described herein are meant to include all racemic mixturesand all individual structural isomers or variants, in particular asdefined by the branch patterns within the HPG structure, or in terms ofthe physical attachment of the surface substituents to the HPG.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sample UPLC chromatograph of docetaxel (DTX).

FIG. 2 shows the peak area of eluents of DTX and its epimer (7-epi-DTX)as a function of time to determine the stability of dHPGs as describedherein incorporating DTX.

FIG. 3A shows a single ion recording (SIR) of DTX and 7-epi-DTX ion(+H⁺) m/z 808.5 in HPG-C_(8/10) incorporating DTX.

FIG. 3B shows a SIR of DTX and 7-epi-DTX ion (+H⁺) m/z 808.5 inHPG-C_(8/10)-MePEG-NH₂ incorporating DTX.

FIG. 4 shows the total ion current (TIC) for HPG-C_(8/10)-MePEG-NH₂incorporating DTX.

FIG. 5 shows a prior art fragmentation pattern proposed for DTX.

FIG. 6 shows the peak area of eluents of DTX (and 7-epi-DTX) as afunction of HPG-C_(8/10)-MePEG-NH₂ incorporating DTX at pH 7.4 and at pH6.0.

FIG. 7A shows percent KU7 cell proliferation as a function ofconcentration of HPG-C_(8/10) for a normal core (NC) formulation and acondensed core (CC) formulation.

FIG. 7B shows percent KU7 cell proliferation as a function ofconcentration of HPG-C_(8/10)-MePEG for a normal core (NC) formulationand a condensed core (CC) formulation.

FIG. 8 shows percent KU7 cell proliferation as a function of polymerconcentration to determine biocompatibility of various dHPGs.

FIG. 9 shows 400 MHz proton (top) and HSQC spectra of HPG-C_(8/10) inD₆-DMSO. D, L₁₃, and L₁₄ represent dendritic, linear 1-3, and linear 1-4units, respectively.

FIG. 10 shows (A) 400 MHz HSQC proton (top) and HSQC spectra ofHPG-C_(8/10)-MePEG_(6.5) and (B) superimposed 400 MHz HSQC spectra ofMePEG 350 epoxide, O/DGE, and HPG-C_(8/10)-MePEG₁₃ polymer.

FIG. 11 shows (A) base-catalyzed ethanolysis of PTX ester linkage togenerate Baccatin III and its side chain ethyl ester(N-benzoyl-3-phenylisoserine ethyl ester) and (B) representativechromatograms illustrating the identification of degradants of PTX by aUPLC-MS/MS assay in a formulation prepared using unpurifiedHPG-C_(8/10)-MePEG.

FIG. 12 shows representative chromatograms illustrating the effect ofpurification of HPG-C_(8/10)-MePEG₁₃ on the chemical stability of PTX(retention time of 2.1 min) measured by UPLC UV analysis. (A)Chromatogram of PTX formulated into unpurified HPG freshly constitutedin PBS (pH 7.4), (B) a chromatogram of the same formulation in (A), aged48 h, and (C) a chromatogram of PTX formulated into purified HPG,freshly constituted in PBS (pH 7.4).

FIG. 13 shows PTX and DTX release from HPG-C_(8/10)-MePEG in artificialurine at 37° C. (A) Cumulative DTX release from HPG-C_(8/10)-MePEG (6and 13 mol). (B) Cumulative PTX release from HPG-C_(8/10)-MePEG inartificial urine (pH 4.6 and 6.5).

FIG. 14 shows confocal fluorescence imaging of KU7 cells illustratingcomplete uptake of HPG-C_(8/10)-MePEG₁₃-TMRCA nanoparticles after a 1 hexposure. (A) Untreated KU7 cells with a DAPI stain allowingvisualization of the nuclei in blue (shown as white in image). (B) KU7cells that have been incubated for 1 h with HPG-C_(8/10)-MePEG₁₃-TMRCAnanoparticles.

FIG. 15 shows in vitro cytotoxicity effects of commercial formulations,Taxol® and Taxotere® and PTX and/or DTX loaded HPG-C_(8/10)-MePEGnanoparticles against KU7-luc cell line, and both low-grade (RT4, MGHU3)and high-grade (UMUC3) human urothelial carcinoma cell lines.

FIG. 16 shows treatment effects of intravesical taxane formulations onorthotopic bladder cancer xenografts. Vehicle controls (PBS & emptyHPG-C_(8/10)-MePEG), Taxol® (1 mg/ml, Bristol-Myers-Squibb), Taxotere®(0.5 mg/ml, Sanofi-Aventis), or paclitaxel (PTX, 1 mg/ml) and docetaxel(DTX, 0.5 mg/ml) loaded into HPG-C_(8/10)-MePEG.

FIG. 17 shows representative sequences of bioluminescence images of micefrom different treatment groups taken on the day of randomization and atdays 18 and 33. Right, representative bladder cross-sections of the samemice in PBS control, Taxotere®, and DTX loaded HPG-C_(8/10)-MePEGtreatment groups.

FIG. 18 shows representative histological sections of bladders harvestedat the end, from mice receiving various treatments: A) PBS, B) Taxol® (1mg/ml), C) HPG-C_(8/10)-MePEG/PTX (1 mg/ml), D) Taxotere® (0.5 mg/ml),E) HPG-C_(8/10)-MePEG/DTX (0.5 mg/ml), F) HPG-C_(8/10)-MePEG (no drug).

FIG. 19 shows (A) one-dimensional proton spectrum (top trace) and HSQCspectrum of HPG-C_(8/10)-MePEG and (B) one-dimensional proton spectrum(top trace) and HSQC spectrum of HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎ acquired ata magnetic field strength of 9.4 T.

FIG. 20 shows mucoadhesive properties of HPGs as assessed by amucin-particle method.

FIG. 21 shows (A) in vitro KU7-luc binding of rhodamine labeled HPGs and(B) cell viability of KU7-luc cells exposed to HPG solutions.

FIG. 22 shows cumulative DTX release from HPG-C_(8/10)-MePEG andHPG-C_(8/10)-MePEG-NH₂ nanoparticles in artificial urine at 6.5.

FIG. 23 shows (A) bioluminescence images of mice from each treatmentgroup except PBS control taken on day 2, 8, and 12 post-tumourinoculation and (B) treatment effects of a single intravesical DTXformulations on orthotopic bladder cancer xenografts. Right panel,detailed view of the treatment arms except the PBS and Taxotere® (0.5mg/ml) groups.

FIG. 24 shows bioluminescence images of mice following a singleintravesical treatment with 0.2 mg/ml of DTX loaded HPG-C_(8/10)-MePEGand HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎.

FIG. 25 shows in vitro cytotoxicity of DTX formulations against theKU7-luc cell line, and both lowgrade (RT4, MGHU3) and high-grade (UMUC3)human urothelial carcinoma cell lines.

FIG. 26 shows treatment effects of single intravesical DTX formulationson orthotopic bladder cancer xenografts. Bioluminescence imaging of miceis shown on the left panel.

FIG. 27 shows representative histological sections of bladders harvestedat the end of study, from mice receiving various formulations of 0.2mg/ml DTX: (A) HPG-C_(8/10)-MePEG-NH₂, (B) HPG-C_(8/10)-MePEG, (C)Taxotere®. Within treatment groups, A, B, and C, numbers 1-3 designatedifferent magnifications.

FIG. 28 shows tumor bioluminescence as a function of time forHPG-C_(8/10)-MePEG incorporating DTX or paclitaxel (PTX) as compared tothe commercial formulations of DTX (Taxotere™) and PTX (Taxol™).

FIG. 29 shows the retention of DTX in the bladder 2 hours afterinstillation of 50 μg of DTX in HPG-C_(8/10)-MePEG orHPG-C_(8/10)-MePEG-NH₂.

FIG. 30 shows orthotopic bladder carcinoma instilled with PBS; freerhodamine (TMRCA); rhodamine labeled HPG-C_(8/10)-MePEG(HPG-C_(8/10)-MePEG-TMRCA), rhodamine labeled HPG-C_(8/10)-MePEG-NH₂(HPG-C_(8/10)-MePEG-NH₂-TMRCA). (B) amount of fluorescence inside thebladder tumors, (C) observed rhodamine fluorescence in tumor tissues asa function of distance from bladder lumen.

FIG. 31 shows (A) ¹H NMR spectrum and (B) ¹³C NMR spectrum ofHPG-C_(8/10)-MePEG-COOH in methanol-d₄.

FIG. 32 shows structural units in HPG polymers. Each dendritic, D,terminal, T, and linear, L₁₃ or L₁₄, unit exists as primary, p, andsecondary, s, unit. For unmodified polymers, R) HPG; for modifiedpolymers, R) HPG, C_(8/10), MePEG, or COOH. The numbering scheme isindicated for the Dp unit.

FIG. 33 shows representative multiplicity-edited HSQC spectra of (A)HPG-C_(8/10)-OH, (B) HPG-C_(8/10)-COOH (high COOH), and (C)HPG-C_(8/10)-MePEG_(6.5). Representative assignments are indicated inthe spectra.

FIG. 34 shows expansions of regions of the HSQC spectra of (A)HPG-C_(8/10)-OH and (B) HPG-C_(8/10)-COOH. Representative assignmentsare given.

FIG. 35 shows FT-IR spectra of HPG-C_(8/10)-MePEG_(6.5) (top),HPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ (center), andHPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ (bottom).

FIG. 36 shows representative structure of PG-C_(8/10)-MePEG-COOH houndto cisplatin.

FIG. 37 shows binding of cisplatin to (empty triangle)HPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ or (filled square)HPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ in distilled water adjusted to pH 6.0.

FIG. 38 shows in vitro release of free cisplatin (empty diamond) orcisplatin bound to (A) HPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ or (B)HPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ at a drug concentration of 1 mg/mL andpolymer concentration of 10 mg/mL. Release media were 1 mM PBS at pHs of4.5 (filled square), 6.0 (empty triangle), 7.4 (inverted triangle), orartificial urine (empty square) at 37° C.

FIG. 39 shows cell viability of KU-7-luc cells after (A) 2 and (B) 72 hof incubation with HPG-C_(8/10)-OH (filled square),HPG-C_(8/10)-MePEG_(6.5) (empty triangle),HPGC_(8/10)-MePEG_(6.5)-COOH₁₁₃ (filled circle), andHPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ (empty diamond).

FIG. 40 shows viability of KU-7-luc cells after (A) 2 and (B) 72 hincubation with free cisplatin (filled circle), cisplatin-loadedHPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ (empty triangle), andHPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ (filled square).

FIG. 41 shows tissue level-depth profiles of DTX in porcine bladdertissue following exposure to 0.5 mg/ml DTX in Tween 80 (filled circle),0.5 mg/ml DTX in HPG-C_(8/10)-MePEG-NH₂ (37 mole amine/mole polymer)(empty square), 0.5 mg/ml DTX in HPG-C_(8/10)-MePEG-NH₂ (10 moleamine/mole polymer) (empty triangle), 0.5 mg/ml DTX inHPG-C_(8/10)-MePEG (filled inverted triangle). Average values forrepeated runs of four formulations, comparing the penetration using HPGpolymer with increasing amine content, compared with Tween 80 (eg theTaxotere formulation). Within each run, 5-6 replicates were run.

FIG. 42 shows tissue level-depth profiles of DTX in porcine bladdertissue following exposure to different DTX formulations with and withoutpre-treatment for 1 hour. 0.5 mg/ml DTX in Tween 80 with chitosanpretreatment (empty circle), 0.5 mg/ml DTX in Tween 80 withHPG-C_(8/10)-MePEG-NH₂ pretreatment (empty diamond). 0.5 mg/ml DTX inHPG-C_(8/10)-MePEG-NH₂ without pretreatment (empty triangle), 0.5 mg/mlDTX in HPG-C_(8/10)-MePEG-NH₂ with chitosan pretreatment (empty invertedtriangle), and 0.5 mg/ml DTX in HPG-C_(8/10)-MePEG with chitosanpretreatment.

FIG. 43 shows AUCs of DTX for different DTX formulations with andwithout pre-treatment for 1 hour. 0.5 mg/ml of DTX in Tween 80 withchitosan pretreatment, 0.5 mg/ml of DTX in Tween 80 withHPG-C_(8/10)-MePEG-NH₂ pretreatment, 0.5 mg/ml of DTX inHPG-C_(8/10)-MePEG-NH₂ without pretreatment, 0.5 mg/ml of DTX inHPG-C_(8/10)-MePEG-NH₂ with chitosan pretreatment, and 0.5 mg/ml DTX inHPG-C_(8/10)-MePEG with chitosan pretreatment. Lines indicate lack ofsignificant difference (p>0.05) between groups in post-hoc Tukeyanalysis after a significant 1-way ANOVA result, p=0.0007. Error barsindicate S.E.M.

FIG. 44 shows tissue level-depth profiles of mitomycin F in porcinebladder tissue following exposure to mitomycin formulations withpre-treatment for 1 hour. Mitomycin F with HPG-C_(8/10)-MePEG-NH₂ (10mol amine/mol HPG) pre-treatment (empty square), and mg/ml mitomycin Fwith HPG-C_(8/10)-MePEG-NH₂ (37 mol amine/mol HPG) pre-treatment (filleddiamond).

FIG. 45 shows SEM images of pig bladders treated ex vivo with HPGdelivery vehicles: controls (Tyrode's buffer, chitosan &HPG-C_(8/10)-MePEG with 0 mol amine/mol polymer).

FIG. 46 shows SEM images of pig bladders treated ex vivo with HPGdelivery vehicles: HPG-C_(8/10)-MePEG-NH₂ 10 & 37 mol amine/mol polymer,0.1, 1 & 10% w/v solution.

FIG. 47 shows an SEM image of the surface of a mouse bladder treatedwith a 2 hour instillation of PBS. The image was taken of a bladderharvested immediately after the 2 hour instillation period.

FIG. 48 shows SEM images of the surface of a mouse bladder treated witha 2 hour instillation of HPG-MePEG 10% solution. The image was taken ofa bladder harvested A) immediately after the 2 hour instillation period,B) 6 and C) 24 h after the instillation.

FIG. 49 shows SEM images of the surface of a mouse bladder treated witha 2 hour instillation of HPG-MePEG-NH₂ (10 mol/mol) 10% solution. Theimage was taken of a bladder harvested A) immediately after the 2 hourinstillation period, B) 6 and C) 24 h after the instillation. Arrowshows loss of a single umbrella cell, exposing lower layers ofepithelium.

FIG. 50 shows SEM images of the surface of a mouse bladder treated witha 2 hour instillation of HPG-MePEG-NH₂ (37 mol/mol) 1% solution. Theimage was taken of a bladder harvested A) immediately after the 2 hourinstillation period, B) 6 and C) 24 h after the instillation.

FIG. 51 shows SEM images of the surface of a mouse bladder treated witha 2 hour instillation of HPG-MePEG-NH₂ (37 mol/mol) 10% solution. Theimage was taken of a bladder harvested A) immediately after the 2 hourinstillation period, B) 6 and C) 24 h after the instillation.

FIG. 52 shows cell counts in urine harvested from mice at the point ofremoving the instillation catheter (2 h, N=6 for all groups) and at thetime of bladder harvest (2, 6, 24 h, n=1-3 for 6 and 24 h samplingtimes).

FIG. 53 shows circulating TNFα levels in mouse blood at 2, 6 and 24 hafter instillation of A) HPG-MePEG 10% solution, B) HPG-McPEG-NH₂ (low)10% solution, C) HPG-MePEG-NH₂ (high) 1% solution, D) HPG-MePEG-NH₂(high) 10% solution, CN) PBS buffer (control), and in U) untreatedanimals. Results are shown with standards used to construct the standardcurve. The dashed line represents the signal of the lowest standard (0.6pg/mL standard concentration).

DETAILED DESCRIPTION

Novel polymers described herein include those shown in Formula I, whichall appear to be related to HPG. Synthesis of HPG has been previouslydescribed, including the production of amphiphilic copolymers andamphiphilic block copolymers, including derivatization with variousfunctional groups and/or the production of copolymers and blockcopolymers (such as the addition of alkyl groups through ester linkagesand the addition of polyalkylene glycol groups). Publications describingpreparation of HPG include: U.S. Pat. No. 5,112,876; U.S. Pat. No.6,469,218; U.S. Pat. No. 6,765,082; U.S. Pat. No. 6,822,068; WO2000/77070; Sunder, A. et al. (1999) Macromolecules 32:4240-46, (2000)Macromolecules 33:309-14, (2000) Macromolecules 33:1330-37, and (2000)Adv. Mater 12:235-239; Knischaka, R. et al., (2000) Macromolecules33:315-20; Haag, R., et al. (2000) Macromolecules 33:8158-66, and (2002)J. Comb. Chem. 4:112-19; Kautz, H., et al. (2001) Macromol. Symp.163:67-73; Karger-Kocsis, J., et al. (2004) Polymer, 45:1185-95; Gao, C.& Yan, D. (2004) Prog. Polym. Sci. 29:183-275; and Tziveleka, L. et al.,(2006) Macromol. Biosci. 6:161-169). Sunder, A. et al., (1999) Angew.Chem. Int. Ed. 38:3552-55 contains a description of the preparation ofamphiphilic modified HPG, as well as derivatization of such polymers,including derivatization with various substituents and functionalgroups.

The dHPGs described herein may include C₁-C₃₀ alkyl chains, or othersimilar alkyl chains. However, the dHPGs described herein may alsoinclude C₁-C₂₀ alkyl chains, or other similar alkyl chains The term“alkyl” is used as it is normally understood to a person of skill in theart and often refers to monovalent saturated aliphatic hydrocarbylgroups having from one to 20 carbon atoms, unless otherwise defined. Thehydrocarbon may be either straight-chained or branched and may containcycloaliphatic or aryl substituents. Alkyl chains may be selected fromone or more of C₁-C₂₀ alkyl chains. Alternatively, the alkyl chains maybe selected from one or more of C₂-C₁₉ or C₃-C₁₈ or C₄-C₁₇ alkyl chains.Alternatively, the alkyl chains may be selected from one or more ofC₅-C₁₆ or C₆-C₁₅ or C₇-C₁₄ alkyl chains. Alternatively, the alkyl chainsmay be selected from one or more of C₈-C₁₃ or C₉-C₁₂ or C₁₀-C₁₅ alkylchains. Alternatively, the alkyl chains may be selected from one or moreof C₅-C₁₅ or C₅-C₁₀ or C₅-C₂₀ alkyl chains. The alkyl chain or chainsselected for the core may depend on the intended use for the dHPG. Forexample, a C₁₈ alkyl did not work as well as a C_(8/10) alkyl forloading paclitaxel.

The HPGs as described herein may include HPGs derivatized withsubstituents having functional groups such that the derivatized HPGs(“dHPGs”) are mucoadhesive and more generally bioadhesive. In the mostgeneral meaning of the term, the dHPGs will form a bond or interact witha biological tissue, which could be a cell or an extracellular material.The bond or interaction may be of any type, including van der Waalsinteractions, hydrogen bonds, electrostatic interactions, ionic bonds orcovalent bonds.

The term “mucoadhesion” or “mucoadhesive” is used as it is normallyunderstood to a person of skill in the art and often refers to anadhesive phenomenon occurring between polymeric materials and thebiological tissue which can include cell surfaces, mucus on cellsurfaces or a mucus-gel layer covering mucosal membranes. As mucin ispresent at the urothelial surface of the bladder, dHPGs containingmucoadhesive functional groups may be used for targeted drug delivery tothe surface of the bladder, as well as to other mucosal membranes.

Generally, the dHPGs described herein have a “core”, which includes andan initiator (for example, trimethyloyl propane (TMP)) and hyperbranchedpolyglycerol. In an embodiment, the hyperbranched polyglycerol core maybe derivatized with C₁-C₂₀ alkyl chains. In an embodiment, the “core”may be enclosed in a “shell”, wherein the shell comprising at least onehydrophilic substituent bound to hydroxyl groups of the core, andwherein the hyperbranched polyglycerol comprises from about 1 to about200 moles of the at least one hydrophilic substituent per mole of thehyperbranched polyglycerol.

“Initiator” as used herein is defined as small molecule comprising analkyl component and more than one, but preferably more than two hydroxylgroups. However, the initiator may have three or four or more hydroxylgroups. An example of an initiator is trimethyloyl propane (TMP).

“Condensed core” as used herein is defined as a core wherein the ratioof C₁-C₂₀ alkyl chains to glycerol units is greater at a centre of thecore compared to a periphery of the core. For example, a C₁₀ alkylchain, may be incorporated into the structure such that it is not evenlydistributed relative to the glycerol throughout the entire hyperbranchedstructure, but rather it is distributed such that it is moreconcentrated in the centre of the hyperbranched core structure (forexample, adjacent the initiator) than near its periphery immediatelyadjacent to the shell substituents. The degree to which the corearchitecture is “condensed” may be controlled by the addition ofreagents. The term “centre” may be defined as being the precise centrehaving a zero volume point. Alternatively, a dHPG may have a radius “r”where the alkyl to glycerol ratio is greater in a central volume havinga radius “rc”, where rc<r, than the alkyl to glycerol overall ratio inthe dHPG as a whole.

For a “regular” or “normal” core, a glycerol epoxide (the hyperbranchingcomponent monomer) and an alkyl epoxide (which imparts the hydrophobicnature to the core) may be added at a constant ratio throughout thereaction. For a condensed core, the alkyl epoxide is added in higherproportion at the earliest stage of the reaction, and is reduced to alower proportion (as low as zero) at the later stages of the reaction.This reduction may occur continuously or occur in discrete steps, therebeing a minimum of two discrete steps.

The core architecture can be defined in terms of the rate of addition ofcomponents. For example, a condensed core polymer can be synthesized inmultiple steps, with each step having a defined ratio of core monomers,one being a glycerol epoxide and the other being a hydrophobic alkylepoxide. A condensed core molecule can be made by having a higher ratioof alkyl epoxides added in earlier step(s) compared to the ratio of thecomponents added in the later or last step(s). Alternatively, the ratiocan be altered over a time course, such that for a first (or earlier)period of time during the reaction a higher ratio of alkyl epoxide toglycerol epoxide is added than is added over later periods of time. Inthis approach, the ratio can be constantly changed as a gradientthroughout the reaction.

The remaining hydroxyl groups of the polymer may be “derivatized” withother hydrophilic substituents such as MePEG or PEG to form ahydrophilic shell, including substituents having hydroxyl, carboxyl,amine (including primary, secondary and tertiary amines) NHS, ether,thiol, halo, thiolether, ester, thioester, amide, succinimides and othersimilar functional groups. During shell formation, it may be possiblefor a portion of the shell substituents to react with hydroxyl groupslocated towards the centre of the polymer. Even if such reactions occur,the core maintains its hydrophobic character.

As used herein, the term “amphiphilic”, or “amphiphilic polymer”, isused as it is normally understood to a person of skill in the art andoften refers to the presence of both a hydrophobic and hydrophilicmoiety in a single molecule. Hydrophobic refers to any substance orportion thereof which is more soluble in a non-polar solvent than in apolar solvent. Hydrophobicity can be conferred by the inclusion ofapolar groups in a molecule, including, but not limited to, long chainsaturated and unsaturated aliphatic hydrocarbon groups and such groupssubstituted by one or more aromatic, cycloaliphatic or heterocyclicgroup(s). Hydrophilic refers to any substance or portion thereof whichis more soluble in a polar solvent than in a non-polar solvent.Hydrophilic characteristics derive from the presence of polar or chargedgroups such as carbohydrates, phosphate, carboxylic, sulfato, amino,sulfhydryl, nitro, hydroxyl and other similar groups. The hydrophilicportion may comprise MePEG, amine, carboxylic acid or NHS.

The term “polyethylene glycol”, or “PEG”, is used as it is normallyunderstood to a person of skill in the art and often refers to suchcompounds having a molecular weight between about 200 to about 20,000,depending on the number of ethylene oxide units in the polymer chain.Preferred molecular weights are from about 200 to about 400, about 200to about 1000 and about 200 to about 2000 although molecular weights ofabout 2000 to about 8000 may also be used.

The term “methoxypoly(ethylene oxide)”, or “MePEG”, is used as it isnormally understood to a person of skill in the art and often refers tosuch compounds having a molecular weight between about 350 to about10,000, depending on the number of ethylene oxide units in the polymerchain. Preferred molecular weights are from about 350 to about 550,about 350 to about 750 and about 350 to about 2000 although molecularweights of about 2000 to about 5000 may also be used.

The phrase “local or targeted delivery” is used as it is normallyunderstood to a person of skill in the art and often refers to deliveryof a compound directly to a target site within an organism.

In some embodiments, the dHPGs as described herein may be used for localor targeted treatment of an indication of the urinary tract (forexample, the urethra and bladder), the digestive tract (for example, themouth, esophagus and colon), the airways (for example, the nose andlungs), the vaginal cavity and cervix and the peritoneal cavity to treatindications such as cancer (for example, bladder, gastric, esophageal,lung, laryngeal, oral, sinus, vaginal or cervical cancers), infection(for example, infections of the digestive tract or the airways), andinflammatory or autoimmune diseases (for example, irritable bladder,inflammatory bowel disease or chronic or acute inflammation) as well asother indications wherein delivery of a drug or other biologicallyactive moiety to a tissue or cell is desired. For example, the dHPGs asdescribed herein may be used for local or targeted treatment ofnon-muscle-invasive bladder cancer. In some embodiments, the polymers asdescribed herein may be used in the preparation of a medicament or acomposition for local or targeted treatment of one or more of theindications listed herein (for example, non-muscle-invasive bladdercancer). Some aspects of this invention make use of compositionscomprising a dHPG described herein and a pharmaceutically acceptableexcipient or carrier. Methods of treating one or more of the indicationslisted herein (for example, non-muscle-invasive bladder cancer) are alsoprovided. Such methods may include administering a dHPG as describedherein or a composition of a dHPG as described herein, or an effectiveamount of a dHPG as described herein or composition of a dHPG asdescribed herein to a subject in need thereof, wherein the dHPGincorporates a biologically active agent.

In some embodiments, the dHPGs as described herein may be used aspre-treatment or co-treatment for increasing drug uptake in a tissue. Insome embodiments, the dHPGs as described herein may be used aspre-treatment or co-treatment for increasing drug uptake of a drug forlocal or targeted treatment of an indication of the urinary tract (forexample, the urethra and bladder), the digestive tract (for example, themouth, esophagus and colon), the airways (for example, the nose andlungs), the vaginal cavity and cervix and the peritoneal cavity to treatindications such as cancer (for example, bladder, gastric, esophageal,lung, laryngeal, oral, sinus, vaginal or cervical cancers), infection(for example, infections of the digestive tract or the airways), andinflammatory or autoimmune diseases (for example, irritable bladder,inflammatory bowel disease or chronic or acute inflammation) as well asother indications wherein delivery of a drug or other biologicallyactive moiety to a tissue or cell is desired. For example, the dHPGs asdescribed herein may be used as pre-treatment or co-treatment toincrease drug uptake of a drug for local or targeted treatment ofnon-muscle-invasive bladder cancer. In some embodiments, the dHPGs asdescribed herein may be used as pre-treatment for increasing drug uptakein a tissue. In some embodiments, the dHPGs as described herein may beused as co-treatment for increasing drug uptake in a tissue. In anembodiment, use of the dHPGs as co-treatment may include where the drugor biologically active moiety is not loaded in the dHPG during treatmentwith the drug or biologically active moiety. In an embodiment, use ofthe dHPGs as co-treatment may include where a portion of or all of thedrug or biologically active moiety is loaded in the dHPG duringtreatment with the drug or biologically active moiety. In someembodiments, the dHPGs as described herein may be used as pre-treatmentand co-treatment for increasing drug uptake in a tissue. In someembodiments, the dHPGs as described herein may be used as pre-treatmentor co-treatment for increasing drug uptake in a tissue as compared todrug uptake in the tissue in the absence of pre-treatment orco-treatment. In some embodiments, the dHPGs as described herein may beused as pre-treatment or co-treatment for increasing drug uptake in atissue without causing necrosis and/or inflammation of the tissue. Insome embodiments, increasing drug uptake in a tissue may include causingloss of umbrella cells. In some embodiments, increasing drug uptake in atissue may include causing loss of umbrella cells without causingnecrosis and/or inflammation of the tissue. In some embodiments, theumbrella cells may be umbrella cells of the urothelial surface of thebladder. The expression “increasing drug uptake” is used as it isnormally understood to a person of skill in the art and often refers toincreasing concentration or accumulation of a drug in a cell or tissue.

In some embodiments, increase in drug uptake may be measured in terms ofincrease in Cavg of drug uptake with use of dHPGs as pre-treatment orco-treatment as compared to Cavg of drug uptake in the absence ofpre-treatment or co-treatment. The person of ordinary skill in the artwill appreciate that Cavg may be measured at different ranges or pointsof tissue depth. For example, Cavg may be measured for 0>3350, 0>1500,200>3350, or 200>1500 μm ranges of tissue depth. In an embodiment, Cavgof drug uptake with the use of dHPGs as pre-treatment or co-treatmentmay be increased by a factor of 1.3 to 4.0, 1.8 to 2.8, 1.3 to 2.4, 2.0to 2.6, 1.5, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, or 4.0fold as compared to Cavg of drug uptake in the absence of pre-treatmentor co-treatment. In some embodiments, increase in drug uptake may bemeasured in terms of increase in Cmax of drug uptake with use of dHPGsas pre-treatment or co-treatment as compared to Cmax of drug uptake inthe absence of pre-treatment or co-treatment. In an embodiment, Cmax ofdrug uptake with the use of dHPGs as pre-treatment or co-treatment maybe increased by a factor of 1.3 to 4.0, 1.8 to 2.8, 1.3 to 2.4, 2.0 to2.6, 1.5, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, or 4.0fold as compared to Cmax of drug uptake in the absence of pre-treatmentor co-treatment. In some embodiments, increase in drug uptake may bemeasured in terms of increase in AUC(x-y) of drug uptake with use ofdHPGs as pre-treatment or co-treatment as compared to AUC(x-y) of druguptake in the absence of pre-treatment or co-treatment. The person ofordinary skill in the art will appreciate that AUC(x-y) may be measuredat different ranges or points of tissue depth. For example, AUC(x-y) maybe measured for 0>infinity, 0>3350, 0>1500, 200>infinity, 200>3350, or200>1500 μm ranges of tissue depth. In an embodiment, AUC(x-y) of druguptake with the use of dHPGs as pre-treatment or co-treatment may beincreased by a factor of 1.3 to 4.0, 1.8 to 2.8, 1.3 to 2.4, 2.0 to 2.6,1.5, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, or 4.0 fold ascompared to AUC(x-y) of drug uptake in the absence of pre-treatment orco-treatment. The person of ordinary skill in the art will appreciatethat there are alternative methods for measuring increase in druguptake, for example, a permeability enhancement ratio, R, calculated asa quotient of permeabilities in the presence and absence ofpre-treatment or co-treatment, as reported in Grabnar et al.(International Journal of Pharmaceutics 256 (2003) 167-173) may be used.

In some embodiments, the dHPGs as described herein may be in the solventaddition form. The dHPGs may be associated with a non-stoichiometricamount of a solvent, water and/or buffers, typically expressed as weightor volume percent. The solvent may be, for example, and withoutlimitation, a pharmaceutically acceptable solvent or other biocompatiblesolvent including ethanol, DMSO, propylene glycol, glycerol, PEG200,PEG300, Transcutol or Solutol.

The embodiments the dHPGs as described herein include all possiblestereochemical alternatives, including those illustrated or describedherein.

In some embodiments, dHPGs as described herein include isomers such asgeometrical isomers having different branch patterns. The dHPGssynthesized by methods disclosed herein are random branching HPGs andwill contain glycerol monomers that are fully reacted, e.g. linked inthree directions, or partially reacted, being linked to another monomerin one or two directions. The presence of each branching architecturemay be confirmed by analytical techniques (for example, 2D NMR HSQCexperiments).

Compositions and dHPGs according to some embodiments described hereinmay be administered in any of a variety of known routes. The dHPGs couldbe administered as an intravesical dosing solution or in othercompositions created to function as a rinse (including an oral rinse, anintraperitoneal irrigation solution or an irrigation for nasal orvaginal cavities), an eyedrop, an oral solution to be swallowed, anaerosol, or a solution for inhalation as a spray, or as a semi-solid tobe inserted into close proximity to a biological tissue such as amucosal surface.

It is understood that it could be potentially beneficial to restrictdelivery of the dHPGs described herein incorporating a drug or otherbiologically active agent to the target tissue or cell to which drugdelivery is desired. For example, it is contemplated that the selectivedelivery of dHPGs as described herein incorporating a biologicallyactive agent to the urothelial surface of the bladder in a subjecthaving or suspected of having non-muscle-invasive bladder cancer mayprovide therapeutic effect without producing significant side effects inother tissues of the body. An example of a method that may be suitablefor the administration of a dHPG as described herein incorporating ataxane is intravesical instillation. Intravesical instillation is alsoan example of a method that may be suitable for administration of a dHPGas described herein for use as a pre-treatment or co-treatment forincreasing drug uptake in a tissue. Intravesical instillation is a meansof drug delivery whereby a solution is inserted into a vesical such asthe bladder. In delivery to the bladder, the solution is typicallyadministered by means of a catheter inserted through the urethra intothe bladder. The solution is instilled and typically retained in thebladder for a period of time such as about 1 or about 2 hours. A typicalvolume of instillation is in the range of about 10 to about 50 mL. Afterthe dwell time, the volume of solution, and any accumulated urine whichhas diluted the solution would be evacuated to end the procedure. Thedwell time represents the time of maximum drug exposure duringintravesical therapy, as the majority of the drug is removed during theevacuation step. Other examples of compositions or methods to facilitatelocalized tissue delivery would be apparent to one of skill in the art.For example, the dHPGs as described herein may be used could be inpharmaceutical compositions wherein the dHPG contains a taxane or otherhydrophobic drug in the core of the dHPG along with a second drug, whichmay also be formulated into the HPG, or the second drug may be combinedin solution with the dHPG for delivery. Furthermore, the dHPGs may becombined with a targeting agent (for example, an antibody to epidermalgrowth factor receptor, which is overexpressed in bladder tumors,Herceptin, or VEGF).

Suitable pharmaceutical compositions may be formulated by means known inthe art and their mode of administration and dose determined by theskilled practitioner. For intravesical instillation, a dHPGincorporating a biologically active agent may be dissolved aninstillation vehicle such as water, a co-solvent system containingwater, an isotonic aqueous solution such as normal saline or dextrose 5%in water, or in a buffered system to control pH at a favorable level,such as about pH 6-8, or another suitable range, e.g. about pH 4-6 orabove pH 8. The pH may be controlled at a specific range to providebenefit in optimizing drug release kinetics, drug stability, maximalmucoadhesion, maximum solubility or a combination thereof. Otherpharmaceutically acceptable vehicles used for administration of awater-soluble drug delivery systems are also contemplated. Manytechniques known to one of skill in the art are described in Remington:the Science & Practice of Pharmacy by Alfonso Gennaro, 20^(th) ed.,Lippencott Williams & Wilkins, (2000).

An “effective amount” of a pharmaceutical composition as used hereinincludes a therapeutically effective amount or a prophylacticallyeffective amount. A “therapeutically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result, such as decreased cancer cellproliferation, increased life span or increased life expectancy. Atherapeutically effective amount of a dHPG incorporating a biologicallyactive agent may vary according to factors such as the disease state,age, sex, and weight of the subject, and the ability of the biologicallyactive agent to elicit a desired response in the subject. Dosageregimens may be adjusted to provide the optimum therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the formulation are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result, such as theprevention or the prevention of the progression of an indication.Typically, a prophylactic dose is used in subjects prior to or at anearlier stage of disease.

It is to be noted that dosage values may vary with the severity of thecondition to be alleviated. For any particular subject, specific dosageregimens may be adjusted over time according to the individual need andthe professional judgment of the person administering or supervising theadministration of the compositions. The amount of composition may varyaccording to factors such as the disease state, age, sex, and weight ofthe subject. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It may be advantageous to formulatecompositions in dosage unit form for ease of administration anduniformity of dosage.

In some embodiments, dHPGs as described herein may be used, for example,and without limitation, in combination with other treatment methods. Forexample, dHPGs as described herein incorporating a biologically activeagent may be used as neoadjuvant (prior), adjunctive (during), and/oradjuvant (after) therapy in combination with other therapies known toone of ordinary skill in the art.

In general, dHPGs as described herein may be used to reduce toxicity.Toxicity of the dHPGs described herein may be determined using standardtechniques, for example, by testing in cell cultures or experimentalanimals and determining the therapeutic index, i.e., the ratio betweenthe LD50 (the dose lethal to 50% of the population) and the LD100 (thedose lethal to 100% of the population). In some circumstances, however,such as in severe disease conditions, it may be necessary to administersubstantial excesses of the compositions. Some dHPGs of this inventionmay be toxic at some concentrations. Titration studies may be used todetermine toxic and non-toxic concentrations. Toxicity may be evaluatedby examining a particular dHPG's or composition's specificity acrosscell lines. Animal studies may also be used to provide an indication ifthe polymer has any effects on other tissues.

The dHPGs as described herein may be administered to a subject. As usedherein, a “subject” may be a human, non-human primate, rat, mouse, cow,horse, pig, sheep, goat, dog, cat, etc. The subject may be suspected ofhaving or at risk for having cancer or other disease associated with atissue having a mucosal surface. Such an indication may be of theurinary tract (for example, the urethra and bladder), the digestivetract (for example, the mouth, esophagus and colon), the airways (forexample, the nose and lungs), the vaginal cavity and cervix and theperitoneal cavity. A cancer (for example, bladder, gastric, esophageal,lung, laryngeal, oral, sinus, vaginal or cervical cancers), an infection(for example, infections of the digestive tract or the airways), or aninflammatory or autoimmune diseases (for example, irritable bladder,inflammatory bowel disease or chronic or acute inflammation) as well asother indications may be desired targets of the dHPGs described hereinfor the delivery of a drug or other biologically active moiety.Diagnostic methods for cancers, infections, and inflammatory orautoimmune diseases are known to those of ordinary skill in the art.

For example, the dHPGs described herein may be used for treatment ofnon-muscle-invasive bladder cancer. The dHPGs described herein may beused for preparation of a medicament for treatment ofnon-muscle-invasive bladder cancer. dHPGs described herein may be usedin a method for treatment of non-muscle-invasive bladder cancer. Themethod may comprise administering to a subject in need thereof aneffective amount of a dHPG described herein incorporating a biologicallyactive agent (for example, a taxane). For example, the dHPGs describedherein may be used as a pre-treatment or co-treatment to increase druguptake of a drug for treatment of non-muscle-invasive bladder cancer.

Methods of preparing or synthesizing dHPGs described herein will beunderstood by a person of skill in the art having reference to knownchemical synthesis principles. For example, WO2006/130978 describessuitable synthetic procedures that may be considered and suitablyadapted for preparing polymers described herein.

A general methodology for chemical preparation of a dHPG is described inthe following non-limiting exemplary scheme:

Step 2, addition of monomers, is further defined by the rate of additionof the monomers over the 22-24 hour period. For condensed core polymers,the rate of addition of the alkyl epoxide is faster at earlier stages ofthe reaction period and the rate of addition of the glycerol epoxide maybe slower at earlier stages. However, adjusting the rate of addition isnot required as long as the ratio of the components favors addition ofthe alkyl component in the earlier stages of the reaction relative tothe later stages.

Various alternative embodiments and examples are described herein. Theseembodiments and examples are illustrative and should not be construed aslimiting the scope of the invention.

EXAMPLES Example 1: Synthesis and Characterization of Derivatized HPGs

All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville,Canada) and used without further purification. All solvents were HPLCgrade from Fisher Scientific (Ottawa, Canada) and used without furtherpurification.

Polymerizations were carried out in a three-neck round-bottom flaskequipped with a mechanical stirrer. The second neck was connected to adual manifold Schlenk line, and the third was closed with a rubberseptum through which reagents were added. A typical polymerizationreaction procedure for HPG-C_(8/10)-MePEG is as follows. The initiatortrimethyloyl propane (TMP) is added to the flask under argon atmospherefollowed by potassium methylate solution in methanol (20 wt %). Themixture is stirred using a magnetic stir bar for 15 minutes, after whichexcess methanol is removed in a vacuum. The flask is kept in an oil bathat 95° C., and glycidol is added dropwise over a period of 12 hoursusing a syringe pump. After completion of the monomer addition, themixture is stirred for an additional 5 hours. Octyl/decyl glycidyl etheris then added and the mixture stirred for 24 hours to form HPG-C_(8/10).To this mixture, MePEG350 is added dropwise aver a period of 12 hoursand then stirred for an additional 5 hours. MePEG is preferred over PEGbecause the methyl group of MePEG protects one end of the monomer suchthat the monomer does not become bivalent, which could result incross-linking between the dHPG molecules. Other protecting groups arealso contemplated, including those that can be removed after thesynthesis. In this fashion the HPG may be prepared with PEG chains onthe surface that may be further modified by the addition of otherchemical groups or biomolecules, including peptides, glycopeptides,proteins and the like. This procedure may be modified to synthesisdifferent HPG's, for example, 1,2-epoxyoctadecane may be added to themixture after addition of glycidol to form HPG-C₁₈.

The product is then dissolved in methanol and neutralized by passing itthree times through a cation exchange column (Amberlite™ IRC-150). Theunreacted octyl/decyl glycidyl ether is removed by extraction withhexane. Methanol is removed and the polymer is dialysed for three daysagainst water using cellulose acetate dialysis tubing (MWCO: 1000 g/mol,Spectrum Laboratories Inc.), with three water changes per day. The drypolymer is then obtained by freeze-drying and heat drying.

This procedure can be modified in order to synthesize condensed coredHPGs in which the alkyl chains are concentrated toward the center ofthe polymer, instead of the regular core dHPG wherein the alkyl chainsare positioned randomly throughout the polymer. The core of the polymeris modified by adding the glycerol epoxide and alkyl monomers to thereaction mixture at different rates and/or in different proportions. Inorder to form a condensed core dHPG, all of the alkyl monomer is addedbefore the glycerol epoxide addition is complete so that the outerportion of the hyperbranched structure does not contain any alkylcomponent. Instead, the alkyl component is located towards the core ofthe HPG.

HPG-C_(8/10)-COOH was synthesized by first preparing HPG-C_(8/10) asdescribed above. Scheme II shows the reaction for the addition ofcarboxylic acid functional groups to HPG-C_(8/10):

Pyridine (50 mL) was added to HPG-C_(8/10) (0.5 g) and stirred rapidlyto dissolve the polymer. Dimethylaminopyridine (0.2 g, 0.0016 moles) wasadded followed by the slow addition of succinic anhydride (12 g, 0.12moles). The reaction was stirred overnight at room temperature(approximately 22° C.). Water was added (100 mL) and the mixture stirredfor 30 minutes. Solvents were removed by rotary evaporation with theperiodic addition of water to enable better evaporation of pyridine byazeotropic distillation. The residue was dissolved in methanol anddialyzed against distilled water for 16 hours using aSpectra/PorDialysis membrane (MWCO: 3500 g/mol). The dialysis medium waschanged four times, each time with a greater methanol concentration. Thefinal composition of the dialysis medium was 70% methanol in distilledwater. The solvent was removed by rotary evaporation and the polymerdried in a vacuum oven overnight.

Scheme III shows the first reaction scheme attempted for the addition ofsuccinimidyl carbonate to HPG-C_(8/10). Briefly, HPG-C_(8/10) was driedunder vacuum at 110° C. and then cooled to room temperature.Acetonitrile and DCM were added to dissolve the polymer.N,N′-disuccinimidyl carbonate (DSC) was then added to the flask. Theflask was evacuated and then purged with argon and the reaction wasallowed to proceed overnight at room temperature after adding pyridine.After the reaction, most of acetonitrile was removed by rotaryevaporation. Methyl tert-butyl ether (MTBE) was added to precipitate thepolymer. The supernatant was decanted and DCM was added to dissolve thepolymer. The material was filtered through a 10-15 μm Buchner funnel toobtain a clear solution, which was rotovapped to remove the DCM. MTBEwas added to precipitate the polymer. The final HPG-C_(8/10)-NHS productwas dried under vacuum at room temperature.

A second synthetic route was attempted after it was shown that the firstattempt produced HPG-C_(8/10)-NHS that was highly reactive, to theextent that it was unstable even when stored at −20° C., resulting incross-linking of the matrix. Scheme IV shows the second reaction schemeattempted for the production of HPG-C_(8/10)-NHS. The synthesis involvesproducing HPG-C_(8/10)-COOH as described above as an intermediate, thenreacting it further with NHS to produce HPG-C_(8/10)-COOH—NHS.

HPG-C_(8/10) and succinic anhydride are dissolved in pyridine andreacted for 24 h at room temperature with dimethylaminopyridine (DMAP)as a catalyst. The reaction was terminated by the addition of an equalvolume of water and the pyridine was removed by rotovapping thesolution. The aqueous solution of HPG-C_(8/10)-COOH was dialyzed for 72h (MWCO: 3500 g/mol) to remove residual solvent, and freeze dried. TheHPG-C_(8/10)-COOH was further reacted with N-hydroxy succiniamide (NHS)for 24 hours at room temperature in dimethyl formamide (DMF) withN,N′-dicyclohexylcarbodiimide (DCC) as the catalyst. At the end of thereaction, DMF was removed through rotary evaporation. The product wasisolated as described above. It was precipitated with MTBE, filtered inacetonitrile, rotovapped and precipitated with MTBE prior to drying.

HPG-C_(8/10)-MePEG-NH₂ batches with various amine densities wereproduced using the procedure below, summarized in Scheme V. Variousstoichiometries of reagents were used for each batch, described in Table1.

HPG-C_(8/10)-MePEG (4 g) was dissolved in 15 ml anhydrous 1,4-dioxane.Potassium hydride (0.45 g) was rinsed with hexanes three times and driedunder vacuum. The polymer solution was combined with the KH and stirredat room temperature until a clear solution was formed, approximately 20%of the OH groups on HPG-C_(8/10)-MePEG were deprotonated.N-(2,3-epoxypropyl)phthalimide) (EPP) (1.184 g) was dried by dissolutionin dichloromethane with stirring overnight over Na₂SO₄ or MgSO₄. Thesolution was filtered and dried under vacuum to remove thedichloromethane. The dried EPP was dissolved in anhydrous 1,4-dioxaneand added to the polymer with stirring overnight at about 85-90° C. Theproduct was neutralized by passing it three times through a cationexchange resin column (Amberlite IRC-150) and then precipitated threetimes from ether to remove unreacted EPP. By NMR, 15.5% of thephthalimide groups were attached to the HPG-C_(8/10)-MePEG. Cleavage ofthe phthalimide function was achieved by hydrazinolysis (refluxing withhydrazine monohydrate). Excess hydrazine monohydrate solution (2 mL) wasadded to the solution of the polymer in methanol and the mixture wasrefluxed for 48 h. After refluxing, the methanol was evaporated, thepolymer was dialysed against water using a MWCO: 10000 g/mol membranefor 48 h and freeze dried.

TABLE 1 Stoichiometry of reagents used to produce HPG-C_(8/10)-MePEG-NH₂HPG-NH₂ information Target NH₂ Mass of reagents (g) substitution %HPG-C_(8/10)-MePEG KH EPP  5% 2 0.1 0.2 15% 4 0.45 1.184 20% 4 0.6 1.575

The obtained polymers have been characterized by NMR, FTIR, DSC and TGA.NMR is particularly useful in confirming the branched structure and thepresence of surface groups added to the shell of the polymer. For somesurface chemistries, FTIR analysis is also useful to confirm theconsumption of hydroxyl groups and their replacement with other groups,wherein the chemistry of those groups provides a distinct IR spectrumfrom the rest of the HPG structure, for example, the addition of C═Obonds. For example, FTIR may be used to confirm the addition of —COOHgroups to the surface or the addition of groups through an esterlinkage.

Example 2: Encapsulation of Paclitaxel or Docetaxel into the dHPGs

Paclitaxel or docetaxel together with a dHPG may be dissolved in a smallamount of acetonitrile and dried in an oven at 60° C. for one hour, thenflashed with a nitrogen stream to eliminate traces of the organicsolvent. The resulting dHPG/paclitaxel or dHPG/docetaxel matrix may behydrated with 10 mM phosphate buffered saline (pH 7.4), vortexed for twominutes and incubated in an oven at 60° C. for one hour. The resultingsolution is generally clear. In those cases where a white precipitationwas observed, the solution may be centrifuged (18 000 g for ten minutes)and the supernatant may be transferred to a new vessel and kept in acool place until use.

Example 3: Stability of Docetaxel and Paclitaxel in dHPGs

The stability of docetaxel (“DTX”) incorporated into dHPGs ischaracterized in terms of the degradation of DTX to inactive breakdownproducts, and its interconversion to its bioactive epimer (“7-epi-DTX”).Formation of the epimer for paclitaxel and docetaxel is known to occuras an equilibrium whereas degradation to inactive breakdown products isirreversible. Stability as described in various dHPGs has been analyzedusing ultra performance liquid chromatography (UPLC). A Waters AcquityUPLC BEH C₁₈ column (2.1×50 mm, 1.7 μm) was used for separation of majordegradation peaks. The injection volume was 3 μL. The mobile phase was a10 mM solution of ammonium acetate that was prepared by weighing 0.385 gof the salt and dissolving it into 500 mL of HPLC grade water. The pHwas adjusted to pH 4.0 using acetic acid. Stock solutions of DTX (2mg/mL) were prepared in methanol and stored in a −20° C. freezer. A setof standards containing DTX were prepared in 50/50 methanol/water over arange of 0.5-100 μg/mL. Limit of detection (LOD) and limit ofquantitation (LOQ) were both 1 μg/mL. The calibration curve from 1-100μg/mL was linear with R² of 0.9998 for DTX. A 1/x weighting was applied.Method accuracy and precision were verified at LOQ (1 μg/mL) andmid-range (10 μg/mL). Five replicate injections were made in each case.Table 2 summarizes the accuracy and precision obtained for DTX at theseconcentrations.

TABLE 2 Accuracy and precision obtained for detection of DTX by UPLCSample Analyte Theoretical Average % RSD # Name Conc. (μg/mL) Conc.(μg/mL) Accuracy n = 5 1 DTX 1.00 1.12 112% 8% 2 DTX 10.0 9.90  99% 0%

Forced degradation of DTX was performed to generate samples forevaluation of method specificity. Degradation of DTX was achieved bypreparing a solution containing 300 μL of methanol, 150 μL of 5%ammonium hydroxide, and 50 μL of a stock DTX prodrug which degrades toDTX within minutes at alkaline pH. DTX prodrug conversion to DTX and DTXdegradation were monitored for over two hours. After 2.2 hours on-tray(room temperature), effectively all prodrug degraded to DTX and othercomponents. The degraded sample was analyzed using the method describedabove to ascertain resolution of DTX prodrug, DTX, and relateddegradants.

Using the above UPLC method, DTX is the eluent at 2.99 minutes and7-epi-DTX is the eluent at 3.15 minutes. A sample chromatograph is shownin FIG. 1. The peaks between 1.4 and 2.0 minutes are products of thedegradation of DTX and 7-epi-DTX. The peak area of DTX and 7-epi-DTX wascalculated as a function of time and then used to determine thepercentage of DTX or epi-DTX remaining in the dHPG. The results areshown in FIG. 2. As can be seen from FIG. 2, those formulationscomprising DTX were stable when the HPG polymer was HPG-C_(8/10) andHPG-C_(8/10)-MePEG which retained over 90% of the incorporated DTX asDTX and 7-epi-DTX over a period of 72 hours in PBS buffered to pH 7.3.The remaining amount of drug had degraded to inactive components andeach of these which contributed more than 2% of the total samples wereidentified by mass spectrometry MRM experiments. These experiments wereconducted to identify the ion fragments associated with DTX's knowndegradation products.

A single ion recording (SIR) of DTX and 7-epi-DTX ion (+H⁺) ink 808.5 inthe HPG-C_(8/10) and HPG-C_(8/10)-MePEG-NH₂ formulations are compared inFIGS. 3A and 3B, respectively. Both chromatograms show the presence ofthe 7-epi degradant, although it is present in greater proportion in theHPG-MePEG-NH₂ sample. For FIGS. 3A and 3B the tallest peaks are DTX andthe lower peaks are 7-epi-DTX. Although the total ion chromatogram showsa very high signal overall due to the polymeric constituents (FIG. 4) inthe formulation, additional masses known to match DTX degradantfragments (Kumar et al 2007 Isolation and characterization ofdegradation impurities in docetaxel drug substance and its formulation,Kumar et al., Journal of Pharmaceutical and Biomedical Analysis 12 Mar.2007 43(4):1228-1235) were identified coinciding with the largestdegradant peaks (FIG. 5). M/z of 226 and 282 were observed at 1.5minutes, which may correspond to fragments from the DTX side chain.However, m/z of 583 was present, which corresponds to 10-deacetylbaccatin III+K⁺, indicating the taxane contains both the core and thesidechain. M/z of 320 and 562 were also observed. The peak at 2 minuteshad m/z=581 and 583, which may correspond to 10-oxo-10-deacetylbaccatinIII+K⁺ and 10-deacetyl baccatin III+K⁺, respectively. This peak positionis also in the region of the chromatogram where baccatin degradants areexpected to be observed.

Stability of some of the formulations may be further increased byadjusting the pH of the formulations. For example, a compositioncomprising HPG-C_(8/10)-MePEG-NH₂ incorporating DTX dissolved in anaqueous medium with buffer salts in a ratio in order to obtain a pH of5.5-6.5 is more stable than the same composition excluding the buffersalts, or a composition with altered buffer salt composition, e.g. a PBSbuffer yielding a pH of 7.4 (FIG. 6). Appropriate buffer salts includephosphate buffering salts. Alternatively, the pH of the compositioncould be lowered by adding an acid such as HCl to the composition.Furthermore, the degradation of DTX is slowed significantly by alteringthe pH of the composition.

Example 4: In Vitro Biocompatibility of dHPGs

The toxicity of the dHPGs was measured by determining whether differentdHPG formulations could kill KU7 cancer cells. These experiments wereconducted using dHPGs that did not incorporate any drug or biologicallyactive moiety. The results are shown in FIGS. 7A and 7B. FIG. 7A showsthe percent KU7 cell proliferation as a function of concentration ofHPG-C_(8/10), for both a regular core formulation and a condensed coreformulation. Changing the core architecture did not affect the stabilityof HPG-C_(8/10) significantly. FIG. 7B shows the viability of KU-7 cellsas a function of concentration of HPG-C_(8/10)-MePEG, for both regularcore formulations and condensed core formulations. Polymers with acondensed core show a 10 times higher IC50 for cell viability ascompared to a regular core polymer. The dHPGs having a condensed coreare better tolerated by cells than dHPGs having a regular core. Bothformulations contained 6.5 mol of MePEG per mol of HPG. The condensedcore formulations were less cytotoxic to the KU-7 cells than the regularcore formulations. Table 3 shows the volume ratios of monomers used toprepare the formulations shown in FIGS. 7A and 7B.

TABLE 3 Volume Ratios of monomers used to prepared formulations shown inFIGS. 7A and 7B Volume Volume Octyl/decyl Reaction Glycidol glycidylether Formulation Step (mL/% v/v) (mL/% v/v) regular core 1 13/59% 9/41% condensed core 1 9/50%  9/50% regular core 1 13/59%  9/41%condensed core 1 9/50%  9/50% 2 4/100% 0/0%  condensed core 1 9/50% 9/50% 2 8/100% 0/0%  regular core 1 13/59%  9/41%

FIG. 8 shows that HPG-C_(8/10) polymers, without a MePEG outer shell areamong the least tolerated, and that without the MePEG shell, alteringthe core from the regular to the condensed core architecture has nobenefit in terms of improved cell viability. HPG-OH signifies that it isa HPG without MePEG on the surface. However, when MePEG is added thebenefit becomes noticeable. The HPG-C_(8/10)-MePEG_(6.5) formula (with6.5 mol MePEG per HPG) shows tolerability comparable to the HPG-C_(8/10)when the normal core versions are compared, but when the condensed corearchitecture is used, the tolerability of the HPG-C_(8/10)-MePEG_(6.5)improves to the level of the regular core HPG-C_(8/10)-MePEG13, whichhas double the amount of MePEG in the shell. Previously disclosedHPG-MePEG polymers (Mugabe C. et al. 2008 BJUI 103:978-986) also hadhigher MEPEG amount, although they were not quantified, they areestimated at >15 mol % and were being well tolerated.

Example 5: In Vitro Cell Uptake Assay Showing that dHPGs are Carriedinto Cells

The uptake of fluorescien-labeled HPG-C_(8/10)-COOH—NHS andHPG-C_(8/10)-COOH into KU7 cells was examined. The dHPG was dissolved inFBS-free media at 1 mg/mL. Into each plate of a 12-well plate, 250 μL ofdHPG was exposed to cells on coverslips. The plate was washed twice withPBS followed by a 10 minute fix with 3.7% formaldehyde in PBS. Again theplate was washed twice with PBS. The coverslips were mounted usingProlong Gold™ with DAPI.

A Z-stack of HPG-C_(8/10)-COOH was also viewed in order to confirm thatthe polymer was actually taken up into the KU-7 cell and did not merelyremain on the surface of the cell. As fluorescence was observed at allangles viewed, it was found that the dHPGs were inside the cell. Theseresults show that the dHPGs are taken up into the cells and do not causeany untoward effects inside the cell. In vitro data shows that when HPGsare exposed to cells, the HPG-C_(8/10)-COOH—NHS and HPG-C_(8/10)-COOHare both taken up by one hour. In the body however, the contact is notas complete as the in vitro scenario and prolonged exposure tofacilitate this uptake is required.

Example 6: Loading of Taxanes into Condensed Core and Regular Core dHPGs

The maximum drug loading of condensed and regular coreHPG-C_(8/10)-MePEG was investigated. DTX and PTX were loaded intoHPG-C_(8/10)-MePEG to target drug concentrations of 0.5, 1.0, 2.0 and3.0 mg/mL. Solutions of 100 mg/mL of polymer in THF were prepared andDTX or PTX were added. The THF was dried under a N₂ stream for about twohours and then dried in a hood oven overnight. TheHPG-C_(8/10)-MePEG/PTX and HPG-C_(8/10)-MePEG/DTX matrices were hydratedwith PBS buffer (pH 7.4). The resulting solutions were spun down at14000 rpm for about 15 minutes. The supernatant liquids were tested byHPLC to obtain the concentrations of drug encapsulated in theHPG-C_(8/10)-MePEG. The results are shown in Table 4.

TABLE 4 Theoretical and actual loading of DTX and PTX into condensedcore (“CC”) and regular core (“RC”) HPG-C_(8/10)-MePEG Actual ActualLoading of Actual Actual Loading DTX into CC Loading of Loading of ofPTX RC HPG- PTX into CC DTX into RC HPG- Target C_(8/10)- HPG-C_(8/10)-HPG-C_(8/10)- C_(8/10)- Loading MePEG MePEG MePEG MePEG (mg/mL) (mg/mL)(mg/mL) (mg/mL) (mg/mL) 0.5 0.47 0.37 0.47 0.45 1.0 0.92 0.67 0.88 0.672.0 1.40 0.80 1.80 0.70 3.0 2.30 0.29 2.30 0.50 It was found thatloading was superior for DTX than for PTX.

Example 7: Synthesis and Characterization of HPG-C_(8/10) andHPG-C_(8/10)-MePEG

Polymerization of octyl/decyl glycidyl ether (O/DGE, C_(8/10)) coremodified HPGs was carried out in a single pot synthetic procedure basedon ring-opening polymerization of epoxides according to reportedprotocols (Kainthan, R. K., Mugabe, C., Burt, H. M., Brooks, D. E.,2008. Biomacromolecules 9, 886-895).

All chemicals were purchased from Sigma-Aldrich (Oakville, ON) and allsolvents were HPLC grade from Fisher Scientific (Ottawa, ON). α-epoxy,ω-methoxy polyethylene glycol 350 (MePEG 350 epoxide) was synthesizedfrom a reaction of MePEG 350, sodium hydroxide, and epichlorohydrin.Octyl/decyl glycidyl ether, potassium methylate and trimethyloyl propane(TMP) were obtained from Sigma-Aldrich and used without furtherpurification.

120 mg of the initiator (TMP) was mixed with 1.5 ml of potassiummethylate solution in methanol (25%, w/v) and added to a three-neckround-bottom flask under argon atmosphere. The mixture was stirred at105° C. for 1 h, after which excess methanol was removed under vacuum,then, 13 ml of glycidyl and 9 ml of O/DGE mixture was injected using asyringe pump at a rate of 1.4 ml/h to the initiator. The stirring ratewas fixed at 68 rpm using a digital overhead stirring system (BDC2002).After completion of monomer addition the mixture was stirred for anadditional 6 h. Purified polymers were obtained by extraction withhexane to remove unreacted octyl/decyl glycidyl ether. The product wasthen dissolved in methanol and neutralized by passing three timesthrough a cation exchange column (Amberlite IRC-150, Rohm and Haas Co.,Philadelphia, Pa.). Methanol was removed under vacuum and an aqueoussolution of the polymer was then dialysed for three days against waterusing cellulose acetate dialysis tubing (MWCO 10,000 g/mol, SpectrumLaboratories), with three water changes per day.

¹H NMR (400 MHz, D₆-DMSO) δ_(H): 0.75-0.82 (—CH₃, TMP); 0.82-0.91(—CH₃-alkyl on O/DGE); 1.16-1.53 (—CH₂—, alkyl on O/DGE); 2.46 (solvent,D₆-DMSO); 3.16-3.80 (—CH and —CH₂—, from HPG core); 4.8 (—OH).

HPG-C_(8/10)-MePEG containing different amounts of MePEG were preparedand designated HPG-C_(8/10)-MePEG_(6.5) and HPG-C_(8/10)-MePEG₁₃ toindicate the amount of MePEG added to the feed (6.5 and 13 mol of MePEGper mole of HPG, respectively). The synthesis was carried out in asimilar fashion as the HPG-C_(8/10) reaction except that differentamounts of MePEG 350 epoxide were added to the reaction mixture in thefinal step of the synthesis. The reaction scheme for the one-potsynthesis of alkyl (R) derivatized HPG-C_(8/10)-MePEG is summarized inScheme VI.

120 mg of the initiator (TMP) was mixed with 1.5 ml of potassiummethylate solution in methanol (25%, w/v) and added to a three-neckround-bottom flask under argon atmosphere. The mixture was stirred at105° C. for 1 h, after which excess methanol was removed under vacuum,then, 13 ml of glycidol and 9 ml of O/DGE mixture was injected using asyringe pump at a rate of 1.4 ml/h to the initiator. After all of themixture of glycidol and O/DGE was injected, the reaction was continuedto about 6 h. Then 0.1 ml of potassium hydride (KH) was added to theflask. The mixture was stirred for 1 h, after which 10 ml or 20 ml ofMePEG 350 epoxide was added as a terminal step in the “one pot”synthesis using a syringe pump at a rate of 1.4 ml/h. The amount ofMePEG 350 was added according to the targeting density on HPGs (i.e 10ml of MePEG350 is for the targeting of 6.5 mol of MePEG on per mole ofHPG). The stirring rate was then increased to 90 rpm and the reactionwas continually carried out at 105° C. for overnight. Any traces ofunreacted octyl/decyl glycidyl ether were removed by extraction withhexane. The product was dissolved in methanol and neutralized by passingit three times through a cation exchange column (Amberlite IRC-150, Rohmand Haas Co., Philadelphia, Pa.). Methanol was removed under vacuum andan aqueous solution of the polymer was then dialysed for three daysagainst water using cellulose acetate dialysis tubing (MWCO 10,000g/mol, Spectrum Laboratories), with three water changes per day toremove unreacted MePEG epoxides. Dry polymer was then obtained byfreeze-drying.

¹H NMR (400 MHz, D₆-DMSO) δ_(H): 0.75-0.82 (—CH₃, TMP); 0.82-0.92(—CH₃-alkyl on O/DGE); 1.15-1.55 (—CH₂—, alkyl on O/DGE); 2.50 (solvent,D₆-DMSO); 3.15-3.80 (—CH and —CH₂—, from HPG core); 3.23 (—OCH₃— fromMePEG), 3.32 (residual water); 4.8 (—OH).

HPG-C_(8/10) (MPG without MePEG chains) was prepared by anionic ringopening multibranching polymerization of glycidol from partiallydeprotonated trimethylol propane (TMP) using potassium methylate.HPG-C_(8/10) has numerous terminal hydroxyl end groups, the number permolecule being roughly equal to the degree of polymerization. TheHPG-C_(8/10) core was derivatized with C_(8/10) alkyl chains to create ahydrophobic core, to allow for loading of drug, for example, taxanes.MePEG chains were linked to hydroxyl groups on HPGs. Since MePEG 350epoxide was added to the polymerization reaction after reaction of theother components, a hydrophilic shell is formed to increase the aqueoussolubility of the HPGs.

NMR experiments were conducted to characterize the structure of the HPGpolymers. The fractions of MePEG and alkyl chains on HPGs were estimatedfrom heteronuclear single quantum coherence (HSQC) NMR experimentsrecorded on a Bruker Avance 400 MHz NMR spectrometer using deuteratedsolvents (Cambridge Isotope Laboratories, 99.8% D). Chemical shifts werereferenced to the residual solvent peak. HSQC spectra were analyzedusing Sparky (T. D. Goddard and D. G. Kneller, Sparky 3, University ofCalifornia, San Francisco). FIGS. 9 and 10 show representative protonand 2D HSQC spectra of HPG-C_(8/10) and HPG-C_(8/10)-MePEG polymers. Allthe peaks were assigned to the structural components of the HPGs, usingthe raw material spectra as the starting reference (FIG. 10B). Theproton NMR spectra were similar to those reported (Kainthan, R. K.,Janzen, J., Kizhakkedathu, J. N., Devine, D. V., Brooks, D. E., 2008.Biomaterials 29, 1693-1704; Kainthan, R. K., Mugabe, C., Burt, H. M.,Brooks, D. E., 2008. Biomacromolecules 9, 886-895). HSQC NMR dataconfirmed the structure of HPGs as hyperbranched polymers with thebranching architectures evident in the spectra (FIGS. 9 and 10). Thefractions of each of the substituents were calculated from the volumeintegrals in HSQC experiments. By comparing the integrals of the MePEGmethoxy-group and the O/DGE methyl group to the integral of the TMP CH₃group, the fractions of O/DGE and MePEG (mol/mol) were calculated foreach HPG polymer. The HSQC data showed the absence of unreacted epoxidemonomers (FIG. 10), which indicates the absence of contamination of thepolymer by unreacted monomers.

Molecular weights and polydispersities of the dHPG polymers weredetermined by gel permeation chromatography with multi-angle laser lightscattering detection (GPC-MALLS). Molecular weights were around 80,000g/mol (Table 5).

The physicochemical characteristics of dHPGs are summarized in Table 5.

TABLE 5 The physical characteristics of HPG-C_(8/10)-OH andHPG-C_(8/10)-MePEG loaded with PTX and DTX Structure by Molecular weightNMR & Polydispersity Thermal properties (mol/mol HPG) M_(w) × Tg Td TgTg HPGs MePEG O/DGE 10⁴ M_(w)/M_(n) (° C.)¹ (° C.)² (PTX)³ (DTX)³HPG-C_(8/10) — 4.7 ND ND −37.5 338 — — HPG-C_(8/10)- 4.0 4.7 7.6 1.01−45.2 341 −68.8 −68.3 MePEG_(6.5) HPG-C_(8/10)- 4.6 4.7 8.3 1.22 −55.4344 −54.9 −58.4 MePEG₁₃ ¹Tg, glass transition taken at midpoint oftransition ²Td, degradation temperature taken at maximum weight loss³PTX and DTX were loaded at the maximum loading capacity ofHPG-C_(8/10)-MePEG M_(w), weight average molecular weight determined bygel permeation chromatography connected to MALLS detector (GPC-MALLS)M_(w)/M_(n) polydispersity ND, not determined

Example 8: Effect of Purification Processes on Thermal Properties ofdHPGs and on Physical and Chemical Stabilities of dHPGs Loaded PTX andDTX

The effect of purification processes on the thermal and degradationproperties of dHPGs was evaluated by differential scanning calorimetry(DSC) and thermogravimetric analysis (TGA). Purified dHPGs are referredto polymers that have been through various purification steps, forexample, extraction with hexane to remove unreacted C_(8/10) alkylchains followed by neutralization through a cation exchange column andthen dialysis.

Thermal analysis was conducted using a TA Instruments DSC Q100 and a TGAQ50. DSC runs were obtained by cycling weighed samples in hermeticsealed aluminum pans through a “heat-cool-heat” cycle at 10° C./min overthe temperature range of −90 to 85° C. TGA runs were conducted at aconstant ramping temperature program (20.0° C./min to 500° C.) with agas flow of 40 ml/min (nitrogen). The real-time weight percentage andTGA chamber temperature were recorded. Analysis of the data wasperformed using TA Universal Analysis 2000 software (Version 4.2E, TAInstruments) to find the onset points. The amount of water content indHPGs was determined by titration using Mettler Toledo DL39 Karl FisherCoulometer equipped with AB104-S balance. Known amount of dHPGs weredissolved in anhydrous methanol and titrated with HYDRANAL®-Coulomatreagent (Sigma). The final results were obtained by subtracting thebackground reading from anhydrous methanol.

HPG-C_(8/10) and HPG-C_(8/10)-MePEG exhibited glass transitions attemperatures decreasing from 38 to 55° C. as the MePEG content increasedfrom 0 (HPG-C_(8/10)) to 4.6 mol MePEG/HPG (Table 5). The purificationprocess was observed to have no effect on Tg values of dHPGs and nosignificant effects were observed on thermal stability. Both purifiedand unpurified dHPGs were stable up to a temperature of 300° C. with noindication of thermal decomposition (Table 6).

The effect of purification processes on physical and chemicalstabilities of PTX and DTX loaded HPG-C_(8/10)-MePEG was also evaluated.Both PTX and DTX were loaded into purified or unpurifiedHPG-C_(8/10)-MePEG and the physical stabilities were evaluated byobserving the onset of drug precipitation from PBS (pH 7.4). Chemicalstabilities were assessed by LC/MS/MS to determine the amounts of PTXand DTX and their degradation products.

The purification processes affected the physical and chemicalstabilities of PTX and DTX loaded dHPGs. Maximum achievable PTX and DTXloadings were greater for unpurified polymers and these formulationswere found to be physically more stable than formulations made withpurified HPGs (Table 6). Samples prepared with unpurified polymer didnot precipitate for several days (>3 d), with taxane loading as high as5% (w/w) whereas those prepared with purified polymer and 5% (w/w)taxane loading precipitated within a few hours, or immediately uponconstitution in PBS (Table 6). However, it was observed that PTX and DTXwere chemically unstable in unpurified dHPGs and large fractions of thetaxanes were degraded during the preparation of the formulations. About75-80% of PTX was degraded immediately following loading in unpurifiedHPG-C_(8/10)-MePEG regardless of whether it was dry (bulk) matrix orafter it was reconstituted in PBS (pH 7.4) (Table 6). However, once inbuffer no further PTX degradation occurred, whereas PTX in the drymatrix continued to degrade over 24 h. FIG. 11B shows a chromatogramfrom a formulation of PTX in unpurified HPG-C_(8/10)-MePEG, with a peakcorresponding to PTX and other peaks resulting from the formation ofseveral degradation products. The chromatogram was obtained from aformulation dissolved in acetonitrile immediately after solvent drying(e.g. in the bulk state, prior to constitution in PBS). Two majordegradants were identified by LC/MS/MS, having m/z values of 587 and854, respectively. Based on these masses and the relative retentiontimes of these peaks, their identities are assumed to be baccatin III(m/z 587, FIG. 11A) and 7-epi-taxol (m/z 854), respectively. Otherdegradation products (peak A & B, FIG. 11B) were also observed inunpurified formulations. Based on their relative retention times, peakareas, and the known degradation mechanisms of taxanes, peak B (FIG.11B) is believed to be baccatin V, which is the 7-epi-baccatin III,while peak A is believed to be 10-deacetylbaccatin III. Taxanes loadedin purified polymers however, exhibited different behavior. PTX wasfound to be chemically stable both in bulk and in solution for severaldays and no major degradants were observed during the preparation of theformulations (Table 6 and FIG. 12C). It was observed that PTX and DTX inunpurified HPGs are quickly degraded and this is believed to be due tothe presence of basic impurities in the unpurified polymers. The mostlikely basic impurities came from the excess of potassium methylate andpotassium hydride added during the synthesis of HPGs. Both potassiummethylate and hydride are strong bases and in combination with theresidual moisture in the polymer would create an environment favorablefor both epimerization at the C7 position and ester cleavage of PTX toproduce baccatin III (or 10-deacetyl baccatin III for DTX) (FIG. 11A).The measurement of the pH of dHPG polymers in distilled water showedthat unpurified polymers had a basic pH while purified polymers had anacidic pH (due to the treatment with Amberlite IRC-150, a cationexchange resin), a more stable environment for taxanes. It has beenreported that the maximum stability of taxanes is in the pH range of 3-5(Dordunoo, S. K., Burt, H. M., 1996. Int. J. Pharm. 133, 191-201; Tian,J., Stella, V. J., 2010. J. Pharm. Sci. 99, 1288-1298). The purifiedpolymers were within this pH range (Table 6), hence the improvedchemical stability of the loaded taxanes. The apparent higher drugloadings and greater physical stability of taxanes loaded in unpurifieddHPG polymers may be explained by the fact that the majority of theloaded drugs were degraded to smaller and more hydrophilic molecules(baccatin III and baccatin V) than the parent taxanes, suggesting thatthese degraded molecules were more effectively loaded into dHPGs.

TABLE 6 Effects of polymer purification on polymer properties, and onthe physical and chemical stability of taxane loaded formulations madewith purified and unpurified HPG-C_(8/10)-MePEG₁₃ Unpurified PurifiedPolymer properties HPG-C_(8/10)-MePEG₁₃ HPG-C_(8/10)-MePEG₁₃ Tg −55.8°C. −55.4° C. Td    310° C.    344° C. Water content (% w/w) 0.326 ±0.001 2.051 ± 0.001 pH (10% aqueous solution) 8.5-9 4.4-4.7 Physicalstability (time to precipitation) with increasing taxane loading (% w/w)¹ PTX (h) DTX (h) PTX (h) DTX (h) 1.0 >77 >72 >12 >72 2.0 >77 >72 1 >483.0 >72 >72 0 24 5.0 >72 >72 0 1 Chemical stability of PTX in “bulk” ²26.2 99.8 formulation (% remaining) (t = 0) Chemical stability of PTX (%remaining) Unpurified Purified (t = 24 h) In “bulk” matrix 15.6 99.4 inPBS pH 7.4 constituted formulation 25.8 98.4 ¹ PTX and DTX loading (%,w/w) in HPG-C_(8/10)-MePEG₁₃, also constituted to an equivalent aqueousconcentration (mg/ml) ² “Bulk” matrix signifies the taxane loadedHPG-C_(8/10)-MePEG₁₃ polymer prepared by solvent evaporation prior toconstitution with PBS buffer. t = 0 for the bulk matrix is immediatelyafter its final preparation step, drying to remove solvent.

Example 9: Effect of MePEG Derivatization on Thermal Properties, SurfaceCharge and Particle Size of dHPGs

Particle size and zeta potential analysis was conducted using a MalvernNanoZS Particle Size analyzer using DTS0012 disposable sizing cuvettesfor each analysis. Polymer solutions at a concentration of 15 mg/ml wereprepared in 1 mM NaCl and filtered with 0.22 μm syringe filter (PALLAcrodisc 13 mm with nylon membrane). Sample acquisition parameters were:angle was 173° back-scatter with automatic attenuation; number of runs11 (10 s/run); dispersant was water at 25° C. (viscosity 0.8872 cP andRI 1.330); Mark-Houwink parameter A=0.428 and K=7.67e-05 cm²/s. dHPGswere assumed to have a similar refractive index as polyethylene glycol(PEG) with a RI=1.460 and absorption 0.01. The final data representedthe average of all the runs.

dHPG particles sizes were consistently less than 10 nm in diameter withthe loading of PTX and DTX having no effect on the size of HPG-MePEG(data not shown). Drug loaded HPGs form extremely small nanoparticles ofless than 10 nm.

The presence of MePEG chains on the surface of HPGs had no significanteffect on the overall surface charge on these nanoparticles as measuredby the zeta potentials as follows: HPG-C_(8/10)=−1.29±0.97 mV;HPG-C_(8/10)-MePEG_(6.5)=−0.92±1.68 mV; HPG-C_(8/10)-MePEG₁₃=0.18±0.16mV.

Effects on the glass transition temperature with increasing MePEGdensity were observed. The Tg decreased from −37.5° C. for theHPG-C_(8/10) polymer to −45.2 and −55.4° C. for theHPG-C_(8/10)-MePEG_(6.5) and HPG-C_(8/10)-MePEG₁₃, respectively (Table5). Loading with PTX or DTX also decreased the Tg (Table 5).

Example 10: Loading, Quantification, and Stability of PTX and DTX indHPGs and Release of PTX and DTX from dHPGs

PTX or DTX and dHPGs were dissolved in 1 ml acetonitrile solution in 4ml vials and dried in an oven at 60° C. for 1 h and flashed withnitrogen to eliminate traces of the organic solvent. The resultingdHPG/taxane matrix was hydrated with 1 ml of 50° C. warm 10 mM phosphatebuffered saline (PBS, pH 7.4) and vortexed for 2 min. The resultingsolutions were generally clear but in cases where white particles wereobserved, the solutions were centrifuged (18,000×g for 10 min) andsupernatants were transferred to new vials.

The amount of PTX and DTX incorporated into HPGs was determined byreversed phase HPLC as previously described (Jackson, J. K., Smith, J.,Letchford, K., Babiuk, K. A., Machan, L., Signore, P., Hunter, W. L.,Wang, K., Burt, H. M., 2004. Int. J. Pharma. 283, 97-109). 100 μl ofdHPG/PTX or DTX solution was dissolved with 900 μl of acetonitrile/water(60:40, v/v) and transferred into HPLC vials (Canadian Life Science,Peterborough, ON). Drug content analysis was performed using a symmetryC18 column (Waters Nova-Pak, Milford, Mass.) with a mobile phasecontaining a mixture of acetonitrile, water, and methanol (58:37:5,v/v/v) at a flow rate of 1 ml/min. Sample injection volumes were 20 μland detection was performed using UV detection at a wavelength of 232nm. HPG-C_(8/10) had a limited aqueous solubility resulting in low drugloading of taxanes (data not shown). The presence of alkyl (C_(8/10))chains in dHPGs is important for loading of hydrophobic drugs, howeverit also significantly reduces their water solubility. To increase thewater solubility of HPGs, MePEG 350 chains were added in the terminalphase of the reaction during the synthesis these molecules. A relativelysmall increase in the amount of MePEG in the dHPGs resulted in increaseddrug loading of HPG-C_(8/10)-MePEG₁₃ for both PTX and DTX. DTX loadingin HPGs was higher than for PTX. dHPGs loaded with DTX showed greaterphysical stability than PTX formulations. Maximum loading of DTX (5%,w/w) was greater than for PTX in HPG-C_(8/10)-MePEG₁₃ (2%, w/w).

The physical and chemical stability of taxane loaded dHPGs wereevaluated. The physical stability was evaluated by visual observation ofclarity of the formulations, where precipitation in less than 24 h wasconsidered a physically unstable formulation. Samples were observedimmediately upon rehydration in PBS (t=0), or after 1, 3, 6, 24, 48 and72 h at room temperature. Chemical stability of PTX and DTX weremonitored by the HPLC method as described above. Degradation productswere identified by mass spectrometry analysis using Waters TQD massspectrometer. The system was operated at an electrospray ion sourceblock temperature of 150° C., a desolvation temperature of 350° C., acone voltage of 45 kV, a capillary voltage of 0.70 kV, extractor voltageof 3 kV, RF voltage of 0.1 kV, a cone gas flow at 25 l/h, a desolvationgas flow at 600 l/h and a collision gas flow at 0.2 ml/min. Themolecules undergo electron spray ionization in the positive ion mode.

PTX and DTX release from dHPGs were determined by a dialysis method. 100mg of dHPGs (HPG-C_(8/10)-MePEG_(6.5) or HPG-C_(8/10)-MePEG₁₃) wereweighed and mixed with 1 mg of PTX or DTX in 1 ml acetonitrile solution,spiked with 15 uCi ³H-DTX or ³H-PTX (15 ul) and dried under nitrogenstream to remove the solvent. Radioactive drugs (³H-DTX or ³H-PTX) wereobtained from Moravek Biochemicals and Radiochemicals (Brea, Calif.).The dHPGs/taxane matrix was hydrated with 2 ml of PBS and transferredinto dialysis bags and dialysed against 500 ml of artificial urine (pH4.5 or 6.5) with shaking at 100 rpm. Dialysis membrane tubing waspurchased from Spectrum Laboratories (Rancho Dominguez, Calif.).Artificial urine was prepared according to the method of Brooks et al.(Brooks, T., Keevil, C. W., 1997. Lett. Appl. Microbiol. 24, 203-206),without the addition of peptone or yeast extract. The pH of the solutionwas adjusted to pH 4.5 and or 6.5 using 0.1M HCl. At different timepoints, the volumes of the dialysis bags were measured and a 10 μlsample was taken for measurement of the remaining radioactivity in thedialysis bags and the entire external release media was exchanged withfresh media to maintain sink conditions. The concentration of ³H-DTX or³H-PTX remaining in the dialysis bag at each time point was determinedby beta scintillation counting (Beckman Coulter Canada, Mississagua,ON). The cumulative percent drug released was calculated by subtractingthe amount of drug remaining at each time point from the initial amountof drug at the beginning of the experiment. The data were expressed ascumulative percentage drug released as a function of time. Datarepresent the mean (SD) of three independent experiments.

The pH of urine is usually acidic but is known to vary over a wide range(pH 4.5-8), therefore the effect of pH on the release profiles PTX andDTX loaded in HPG-C_(8/10)-MePEG was evaluated. The release profiles oftaxanes from HPGs were characterized by a continuous controlled releaseand little or no burst phase of release followed by a slowersustained-release phase. DTX was released more rapidly than PTX (75% vs50% drug release after 2 days) from HPG-C_(8/10)-MePEG and almost allDTX released in 6-7 days, compared to 12-14 days for PTX. This wasbelieved to be due to the greater hydrophilicity of DTX whereas morehydrophobic PTX may have greater compatibility and interactions with thealkyl chains (C₈/C₁₀) of the HPG core leading to a slower drug releaserate. Increases in the density of MePEG on HPGs were observed to have noeffect on drug release (FIG. 13A). Changes in the pH of the releasemedium (pH 4.5-6.5) were observed to have no effect on drug release fromHPG-C_(8/10)-MePEG nanoparticles (FIG. 13B). Evaluation of releaseprofiles of PTX and DTX from the HPG nanoparticles using various kineticmodels (including first order, higuchi, and korsmeyer model) indicatedthat both the first order and the higuchi kinetics provided the best fitwith r²=0.98-0.99 (data not shown). There was no statistical differencebetween the formulations in terms of rate of drug release (data notshown).

Example 11: Rhodamine Labeling of HPG-C_(8/10)-MePEG₁₃ and CellularUptake of Rhodamine-Labeled HPG-C_(8/10)-MePEG₁₃

HPG-C_(8/10)-MePEG₁₃ was covalently labeled withtetramethylrhodamine-5-carbonyl azide (TMRCA) according to the method ofHuang et al. with slight modifications (Huang, S. N., Phelps, M. A.,Swaan, P. W., 2003. J. Pharmacol. Exp. Ther. 306, 681-687). 500 mg ofHPG-C_(8/10)-MePEG₁₃ was dissolved in 5 ml of anhydrous 1,4-dioxane. Anappropriate amount of TMRCA was dissolved in anhydrous 1,4-dioxane togive a final concentration of 1 mg/ml. An aliquot of 675 μl of thisfluorescent probe, which corresponds to approximately 20 mol % of HPG,was added to the HPG-C_(8/10)-MePEG₁₃ solution and heated at 80° C. inoil bath under nitrogen stream with stirring for 5 h. The solution wasdialysed against DMF (MWCO 12,000-14,000) until the dialysate wascolourless and then dialysed against distilled water for 24 h. Thefluorescent-labeled polymer (HPG-C_(8/10)-MePEG₁₃-TMRCA) wasfreeze-dried and stored at −80° C. in amber vials.

KU7 cells were allowed to grow on several microscope 1 cm×1 cm coverslips on the bottom of a 10 cm Petri dish until a confluence of ˜75% wasreached which corresponds to a cell number of approximately 7×10⁴ cells.These cell-containing cover slips were washed with warmed PBS threetimes and then placed on parafilm-lined petri dishes with the cell sideup. 250 μl of HPG-C_(8/10)-MePEG₁₃-TMRCA solution (1 mg/ml dissolved inDulbecco's Modified Eagle Medium (DMEM)) were added to the cover slips.Cells were incubated with HPG-C_(8/10) MePEG₁₃-TMRCA for 1, 4, 8, and 24h. For controls, the KU7 cells were incubated in DMEM without anysupplementation. The cover slips were then washed four times vigorouslywith PBS buffer, excess PBS gently blotted and 250 μl of 3.7%paraformaldehyde added to fix the cells for 10 min. Cover slips werewashed an additional three times with PBS and submerged in water. Afterblotting excess liquid, the cells were stained with Prolong® Goldantifade reagent with DAPI (Molecular Probes, Invitrogen) and the slipsmounted cell side down on microscope glass slides. The edges of thecover slips were sealed by clear nail varnish to avoid drying. Thesamples were incubated in the dark overnight to ensure the properstaining of the cells. Samples were observed under an Olympus FV-1000inverted confocal microscope equipped with DAPI (λ_(ex) 340-380 nm;λ_(em), 435-485 nm; dichroic splitter, 400 nm) and rhodamine (λ_(em)530-560 nm; λ_(em), 590-650 nm; dichroic splitter, 570 nm) filters.Direct contrast (DIC) was also performed to visualize cell membranes andwas activated with a 405 nm laser. In order to clearly show that thelabeled polymer was inside the cell, images were analyzed byfluorescence and DIC.

Cellular uptake of rhodamine-labeled HPG-C_(8/10)-MePEG₁₃(HPG-C_(8/10)-MePEG₁₃-TMRCA) was visualized by confocal microscopy ofKU7 cells. After one hour of incubation there was evidence of the uptakeof HPG-C_(8/10)-MePEG₁₃-TMRCA into KU7 cells. Representative images ofthe uptake of HPG-C_(8/10)-MePEG₁₃-TMRCA at 1 h are shown in FIG. 14.Panel A shows untreated KU7 cells with a DAPI stain, which allowsvisualization of the nuclei in blue (shown as white in image). The imageis an overlay of a direct contrast signal (which shows the contour ofthe cell) and the fluorescence signals, which shows the nucleus (white),and the absence of any other fluorescence. Panel B shows KU7 cells thathave been incubated for 1 h with HPG-C_(8/10)-MePEG₁₃-TMRCAnanoparticles. The presence of HPG-C_(8/10)-MePEG₁₃-TMRCA in thecytoplasm is shown by the red fluorescence of the polymer around thenucleus (stained blue with DAPI) (red fluorescence shown as whiteportion in image. Red fluorescence surrounds nucleus shown as darkportion in image). The z-stack of the same cell population from panel B(z-stack image not show), demonstrated that the red fluorescentnanoparticles are present throughout the cytoplasm, rather than beingonly adhered to or present in cell membrane. These nanoparticlesappeared to be distributed uniformly in the cytoplasm, although somepunctate structures were observed indicating thatHPG-C_(8/10)-MePEG₁₃-TMRCA nanoparticles were packaged into smallvesicles for cellular trafficking. There was no fluorescence from thepolymer detected in the nuclear compartment of the KU7 cells.HPG-C_(8/10)-MePEG₁₃-TMRCA nanoparticles have no effect on the viabilityand prevalence of the KU7 cells when compared to the control cells atall time points, indicating that these nanoparticles were highlybiocompatible with this cell line. HPG-C_(8/10)-MePEG₁₃-TMRCAnanoparticles were taken up into KU7 cells by 1 h of incubation andthere were no differences in the images obtained at 1, 4, 8 or 24 h timepoints.

Example 12: In Vitro Cytotoxicity Studies of HPG-C_(8/10)-MePEG

HPG-C_(8/10)-MePEG was prepared according to reported protocols(Kainthan R K, Mugabe C, Burt H M, Brooks D E. Biomacromolecules, 9(3),886-895 (2008)) as described above. ¹H NMR (400 MHz, D₆-DMSO) δH:0.75-0.82 (—CH₃, TMP); 0.82-0.92 (—CH₃-alkyl on O/DGE); 1.15-1.55(—CH₂—, alkyl on O/DGE); 2.50 (solvent, D₆-DMSO); 3.15-3.80 (—CH and—CH₂—, from HPG core); 3.23 (—OCH₃— from MePEG), 3.32 (residual water);4.8 (—OH).

The fractions of MePEG and alkyl chains on HPGs were estimated fromheteronuclear single quantum coherence (HSQC) NMR experiments. Chemicalshifts were referenced to the residual solvent peak. Molecular weightsand polydispersities of the polymers were determined by gel permeationchromatography with multi-angle laser light scattering detection(GPC-MALLS): molecular weight=83,000 g/mol with a polydispersity of 1.22(data not shown).

Particle size analysis was conducted using a Malvern NanoZS ParticleSize analyzer. Drug loaded HPG-C_(8/10)-MePEG formed nanoparticles ofless than 10 nm (7.5±3.4 to 7.8±2.7 nm, data not shown).

PTX or DTX loaded dHPGs were prepared by dissolving PTX (1 mg) or DTX(0.5 mg) and HPG-C_(8/10)-MePEG (100 mg) in 1 ml acetonitrile solutionin 4 ml vials and dried in an oven at 60° C., for 1 h and flashed withnitrogen stream to eliminate traces of the organic solvent. Paclitaxel(PTX) powder was obtained from Polymed Therapeutics, Inc. (Houston,Tex.). Docetaxel (DTX) powder was obtained from Natural PharmaceuticalsInc. (Beverly, Mass.). The resulting HPG-C_(8/10)-MePEG/taxane matrixwas hydrated with 1 ml of 10 mM phosphate buffered saline (PBS, pH 6)and vortexed for 2 min. The amount of PTX and DTX incorporated inHPG-C_(8/10)-MePEG were determined by reversed phase HPLC, PTX and DTXcan be loaded with high drug loadings (maximum loading of 2 and 5% wiw,respectively) by the solvent evaporation method.

Cytotoxic effects of commercial formulations, Taxol® and Taxotere® andPTX and/or DTX loaded HPG-C_(8/10)-MePEG formulations against theKU7-luc cell line, and both lowgrade (RT4, MGHU3) and high-grade (UMUC3)human urothelial carcinoma cell lines were evaluated. Taxol® was fromBristol-Myers-Squibb (Princeton, N.J.). Taxotere® was purchased fromSanofi-Aventis Canada Inc. (Laval, Quebec). The human bladder cancercell lines RT4 and UMUC3 were purchased from the American Type CultureCollection. Cells were maintained in McCoy's medium (Invitrogen,Burlington, ON) containing 10% heat-inactivated fetal bovine serum andkept at 37° C. in a humidified 5% CO₂ atmosphere. MGHU3 cells wereobtained as a generous gift from Dr. Y. Fradet (L'Hotel-Dieu de Quebec,Quebec, Canada) and maintained in MEM supplemented with 10% fetal bovineserum and 2 mM L-glutamine (Invitrogen). KU7 was kindly provided by Dr.C. Dinney (M D Anderson Cancer Center, Houston, Tex., USA) andmaintained in DMEM containing 5% fetal bovine serum. For visualizationpurposes, KU7 cells were infected with a lentivirus containing thefirefly luciferase gene by Dr. Graig Logsdon (M. D. Anderson CancerCenter, Houston, Tex., USA), and these subclones were named KU7-luc aspreviously reported (Hadaschik B A, Black P C, Sea J C et al. BJU Int,100(6), 1377-1384 (2007)). Cells were plated at 5,000 cells/well in96-well plates in a 100 μl volume of McCoy's Medium supplemented with10% FBS and allowed to equilibrate for 24 h before freshly preparedsolutions of Taxol®; Taxotere®; PTX loaded HPG-C_(8/10)-MePEG; or DTXloaded HPG-C_(8/10)-MePEG were added. Cells were exposed to the drugformulations for 2 h, to simulate the current clinical standard forinstillation therapy, and cell viability was determined after 72 h usingthe CellTiter96 AQueous Non-Radioactive Cell Proliferation (MTS) assay(Promega, Madison, Wis.) as reported previously (Hadaschik B A, ter BorgM G, Jackson J et al. BJU Int, 101(11), 1347-1355 (2008)). Eachexperiment was repeated three times and MTS values fell within a linearabsorbance range for all cell lines.

All formulations resulted in concentration-dependent inhibition of theproliferation of all cell lines tested (FIG. 15). DTX formulations weremore cytotoxic than PTX formulations although there were no significantdifferences between groups (P>0.05, one-way ANOVA). The IC₅₀ ofTaxotere® was about two- to five-fold lower than that of Taxol®. PTX andDTX loaded HPG-C_(8/10)-MePEG nanoparticles were found to be ascytotoxic as the commercial formulations of Taxol® and Taxotere®,respectively (FIG. 15). Control HPG-C_(8/10)-MePEG nanoparticles (nodrug) showed no cytotoxicity across the concentration range of 15-1,500nM (data not shown), while Cremophor-EL® and Tween 80 have been shown tobe toxic to cells even at low concentrations (Iwase K, Oyama Y,Tatsuishi T et al. Toxicol Lett, 154(1-2), 143-148 (2004); Henni-SilhadiW, Deyme M, Boissonnade M M et al. Pharm Res, 24(12), 2317-2326 (2007)).

Example 13: Efficacy of Intravesical PTX and DTX Formulations inOrthotopic Model Bladder Cancer

In vivo studies were done in a total of 60 nude mice to evaluate theefficacy of intravesical Taxol® (1 mg/ml, Bristol-Myers-Squibb);Taxotere® (0.5 mg/ml, Sanofi-Aventis); PTX (1 mg/ml) loadedHPG-C_(8/10)-MePEG; and DTX (0.5 mg/ml) loaded HPG-C_(8/10)-MePEG in amouse xenograft model of bladder cancer. This orthotopic mouse model hasbeen reported (Mugabe C, Hadaschik B A, Kainthan R K et al. BJU Int,103(7), 978-986 (2009); Hadaschik B A, Black P C, Sea J C et al. BJUInt, 100(6), 1377-1384 (2007); Hadaschik B A, ter Borg M G, Jackson J etal. BJU Int, 101(11), 1347-1355 (2008)). Animal studies were carried outin accordance with the Canadian Council on Animal Care. Eleven week oldfemale nude mice (Harlan, Indianapolis, Ind.) were anaesthetized withisoflurane. A superficial 6-0 polypropylene purse-string suture wasplaced around the urethral meatus before a lubricated 24 G Jelcoangiocatheter (Medex Medical Ltd., Lancashire, UK) was passed throughthe urethra into the bladder. After a single irrigation of the bladderwith PBS, two million KU7-luc cells were instilled as a single cellsuspension in 50 μl and the purse-string suture was tied down for 2.5 h.To quantify in vivo tumour burden, animals were imaged in supineposition 15 min after intraperitoneal injection of 150 mg/kg luciferinon days 4, 11, 18, 25, and 33 with an IVIS200 Imaging System(Xenogen/Caliper Life Sciences, Hopkinton, Mass.). Data were acquiredand analyzed using Living Image software (Xenogen). On day fivepost-tumour inoculation, mice were randomized to receive a 50 μlintravesical treatment with PBS (control); HPG-C_(8/10)-MePEG (no drug);Taxol® (1 mg/ml); PTX (1 mg/ml) loaded HPG-C_(8/10)-MePEG; Taxotere®(0.5 mg/ml); DTX (0.5 mg/ml) loaded HPG-C_(8/10)-MePEG. Intravesicaltherapy was given on day 5 and 19 post-tumour inoculation. Levels ofbioluminescence were equivalent among the groups; however, as tumoursvaried between individual mice, for statistical analyses, tumourbioluminescence after treatment was normalized against the initial fluxon day four in each mouse. Necropsy was performed on day 33 after tumourinoculation. The whole bladders were removed, fixed in 10% bufferedformalin and embedded in paraffin. 5 μm sections were prepared andstained with H&E using standard techniques. All slides were reviewed andscanned on a BLISS microscope imaging workstation (Bacus LaboratoriesInc., Lombard, Ill.). After intravesical inoculation of KU7-luc cancercells, all mice developed bladder tumours. However, two mice did notrecover from anaesthesia and died on the same day of tumour inoculation.One mouse in the Taxotere® arm was found dead on the last day of imaging(day 33 post-tumour inoculation) this mouse had the largest tumour inthe group on the previous measurements. Another mouse in DTX loadedHPG-C_(8/10)-MePEG was euthanized due to irreversible weight loss (15%weigh loss). For statistical analysis, these mice were excluded from thestudy. Overall, intravesical PTX and DTX, either the commercial Taxol®and Taxotere®, or the HPG-C_(8/10)-MePEG formulations were welltolerated by mice and no major toxicities or weight losses wereobserved. Intravesical therapy was given on day 5 and 19 post-tumourinoculation. Compared with control mice (PBS & emptyHPG-C_(8/10)-MePEG), PTX and DTX loaded HPG-C_(8/10)-MePEG significantlyinhibited tumour growth (P<0.001, 2-way ANOVA, Bonferroni post-tests)(FIG. 16). Unlike Taxotere®, Taxol® (1 mg/ml) significantly decreasedthe tumour growth (P<0.01, 2-way ANOVA, Bonferroni post-tests) whencompared to the control groups (PBS & empty HPG-C_(8/10)-MePEG).However, no significant difference was observed between Taxotere® andTaxol® treatment arms. Intravesical instillation doses of PTX (1 mg/ml)and DTX (0.5 mg/ml) were chosen based on a previous report with PTX(Mugabe C, Hadaschik B A, Kainthan R K et al. BJU Int, 103(7), 978-986(2009)) and the in vitro cytotoxicity data demonstrating DTX to be morepotent than PTX (FIG. 15). At the end of the study, tumour growth inboth PTX and DTX loaded HPG-C_(8/10)-MePEG nanoparticles was inhibitedby 87 and 97%, respectively, compared to the PBS control group.Taxotere® and Taxol® exhibited a 43 and 65% tumour growth inhibition,respectively. Representative bioluminescence images of mice over time ineach treatment group are shown in FIG. 17. Histological examination ofbladder tissues show that KU7-luc tumours exhibited an aggressive growthpattern and frequent multifocality, but after 33 days post-tumourinoculation, most of the tumours in the treatment arms were generallyconfined to the lamina propria and correlated with high-grade pT1 stagedisease (FIG. 18).

It is speculated that the low nanometer size range of HPGs may permitpenetration between mucin chains and contact the umbrella cells of theurothelium leading to enhanced endocytosis of these nanoparticles intothe bladder wall including tumour tissues. It is also possible that thesurface MePEG chains on the HPGs might interact with mucin glycoproteinsthrough chain entanglement resulting in entrapment of thesenanoparticles in the mucin layers leading to prolonged residence time ofthe drug loaded nanoparticles in the bladder. Table 7 shows that PTX andDTX loaded HPG-C_(8/10)-MePEG nanoparticles exhibited a 2-3-fold higherbladder tissue accumulation than the commercial formulations of Taxol®or Taxotere®.

Example 14: Pharmacokinetics

To evaluate the pharmacokinetic properties of intravesical PTX and DTXformulations, mice were instilled with either Taxol® (1 mg/ml, n=3);Taxotere® (0.5 mg/ml, n=4); PTX loaded HPG-C_(8/10)-MePEG (1 mg/ml,n=4); and/or DTX loaded HPG-C_(8/10)-MePEG (0.5 mg/ml, n=4). Tail bloodsamples were taken at 0, 30, and 60 min post intravesical instillation.During this period mice were still anaesthetized with isoflurane. After2 h, all mice were killed using CO₂ asphyxiation and additional bloodwas removed by cardiac puncture. Blood samples were centrifuged inmicro-haematocrit tubes (Fisher Scientific, Pittsburg, Pa.) orserum-separator tubes (Becton Dickinson) and the serum was snap-frozenin liquid nitrogen. Urine and bladder of each mouse were also harvestedand before freezing, the bladders were cut open to expose the lumen andwere vigorously washed in five sequential 10 ml PBS washes. All sampleswere stored at −80° C. The UPLC-MS/MS system used for analysis consistedof an integrated Waters Acquity UPLC separation system coupled to a massspectometry analysis using Waters TQD mass spectrometer. The system wasoperated at an electrospray ion source block temperature of 150° C., adesolvation temperature of 350° C., a cone voltage of 14 V, a capillaryvoltage of 0.70 kV, extractor voltage of 3 kV, RF voltage of 0.1 kV, acone gas flow at 25 l/h, desolvation gas flow at 600 l/h and a collisiongas flow at 0.2 ml/min. The molecules undergo electron spray ionizationin the positive ion mode. DTX was extracted from the mouse serum bysolvent/solvent extraction method. 50 μl aliquots of the mouse plasmaand standards were mixed with 150 μl of 0.1% formic acid in acetonitrilein a 96-well plate and vortexed for 1 min at room temperature. Thesamples were centrifuged at 5,500 rpm (Allegra™ 25 R centrifuge,Beckman-Coulter) for 10 min at 4° C. Then 100 μl of the supernatant wasmixed with 50 μl of distilled water, mixed and vortexed for 30 s.Bladder tissues were weighed and homogenized in 0.1% formicacid/methanol using zirconia beads (Biospec Products) and mini-beadbeater equipped with microvial holder (Biospec Products) for 60 s. Thesamples were centrifuged at 14,000 rpm (Allegra™ 25 R centrifuge,Beckman-Coulter) for 2 min at 4° C. 150 μl of 0.1% trifluoroacetic acidin methanol was added to the samples, mixed and vortexed at 14,000 rpm(Allegra™ 25 R centrifuge, Beckman-Coulter) for 15 min at 4° C. Allsample analyses were performed using UPLC-MS/MS. The limit ofquantification for DTX was 10 ng/ml with a recovery of 97% from spikedcontrol samples.

Several serum samples had non-quantifiable or no detectable levels. Ingeneral, serum levels of both PTX and DTX were low (5-20 ng/ml)following intravesical instillation. There were no significantdifferences (P>0.05) in serum levels between different groups and/or atdifferent time points (Table 7). However, bladder tissue levels wereabout 100-500-fold higher than the serum levels. PTX and DTX loadedHPG-C_(8/10)-MePEG nanoparticles exhibited a 2-3-fold higher bladdertissue accumulation than the commercial formulations, although, thedifferences were not statistically significant (P>0.05, 1-way ANOVA,Bonferroni's multiple comparison test). The final drug concentrations inthe urine were about 3-5-fold lower than the initial dosing solutions.This was due to the urine dilution during the 2 h period of intravesicalinstillation. However, there was no significant difference (P>0.05,1-way ANOVA) in the final urine concentrations of PTX and DTX betweendifferent treatment groups. In general, serum levels of both PTX and DTXwere low (5-20 ng/ml) following intravesical instillation. There were nosignificant differences (P>0.05) in serum levels between differentgroups and/or at different time points (Table 7).

TABLE 7 Pharmacokinetics of intravesical PTX and DTX formulations inorthotopic xenografts Taxane formulations C_(urine) ¹ C_(bladder) ²C_(serum) ³ (ng/ml) (No. of mice) (μg/ml) (μg/g) 1 h 2 h Taxol ® (3)303.2 ± 2.93 ± 8.11 5.49 101.7 0.69 Taxotere ® (4) 134.3 ± 1.22 ± 13.97± 16.02 79.2 0.88 4.8 PTX/HPG-C_(8/10)-MePEG 188.4 ± 7.38 ± 16.02 19.36± (4) 38.6 4.16 14.10 DTX/HPG-C_(8/10)-MePEG 180.4 ± 3.60 ± 9.47 13.74 ±(4) 60.5 1.07 3.0 ¹Final concentration in mouse urine after 2 h ofintravesical instillation measured by HPLC ²Concentration of PTX or DTXin mouse bladder tissue following a 2 h intravesical instillationmeasured by LC/MS/MS ³Concentration of PTX or DTX in mouse serum takenat 1 and 2 h post-intravesical instillation measured by LC/MS/MS Datashown are the mean ± SD

Example 15: Synthesis of HPG-C_(8/10)-MePEG and HPG-C_(8/10)-MePEG-NH₂

HPG-C_(8/10)-MePEG was prepared according to the protocol described inExample 7. ¹H NMR (400 MHz, D₆-DMSO) δH: 0.75-0.82 (—CH₃, TMP);0.82-0.92 (—CH₃-alkyl on O/DGE); 1.15-1.55 (—CH₂—, alkyl on O/DGE); 2.50(solvent, D₆-DMSO); 3.15-3.80 (—CH and —CH₂—, from HPG core); 3.23(—OCH₃— from MePEG), 3.32 (residual water); 4.8 (—OH).HPG-C_(8/10)-MePEG-NH₂ with various target amounts of amine substitutionwere synthesized using the procedure below. The amounts of reagents usedare summarized in Table 8. Target amine substitutions represent thetarget number of moles of NH₂ per mole of HPG and are denoted byHPG-C_(8/10)-MePEG-NH_(2(X)), where x is 61, 121, and 161 moles of NH₂per mole of HPG. HPG-C_(8/10)-MePEG was dissolved in 15 ml of anhydrous1,4-dioxane. Potassium hydride (KH) was rinsed with anhydrous hexanethree times to remove the mineral oil and dried under vacuum. Thepolymer solution was combined with KH and stirred at room temperatureuntil a clear solution was formed. N-(2,3-epoxypropyl)-phthalimide)(EPP) (Sigma-Aldrich) was dried by dissolution in dichloromethane withstirring overnight over Na₂SO₄ or MgSO₄. The solution was filtered anddried under vacuum to remove the dichloromethane. The dried EPP wasdissolved in anhydrous 1,4-dioxane and added to the polymer withstirring overnight at 85-90° C. The product was neutralized by passingit three times through a cation exchange resin column (AmberliteIRC-150) and then precipitated three times from ether to removeunreacted EPP. Cleavage of the phthalimide function was achieved byhydrazinolysis with hydrazine monohyhdrate. Excess hydrazine monohydratesolution (2 ml) was added to the solution of the polymer in methanol andthe mixture was refluxed for 72 h. After refluxing, the methanol wasevaporated, the polymer was dialysed against water using a 10,000 MWCOmembrane for 48 h and freeze dried. ¹H NMR (400 MHz, D₆-DMSO) δH:0.75-0.82 (—CH₃, TMP); 0.82-0.92 (—CH₃-alkyl on O/DGE); 1.15-1.55(—CH₂—, alkyl on O/DGE); 2.50 (solvent, D₆-DMSO); 2.60-2.80 (—CH₂—NH₂)3.15-3.80 (—CH and —CH₂—, from HPG core); 3.23 (—OCH₃— from MePEG).Reaction scheme for the surface modification of some of the hydroxylgroups (10-20%) on HPG-C_(8/10)-MePEG polymer withN-(2,3-epoxypropyl)-phthalimide) (EPP) followed by cleavage of thephthalimide functional groups by hydrazinolysis to produceHPG-C_(8/10)-MePEG-NH₂ is summarized in Scheme VII (R, represent thehydrophobic core based on mixture of alkyl (C₈/C₁₀) chains. ( )7,represent the hydrophilic shell based on MePEG 350).

HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎ was selected for drug loading and furtherevaluation in animal studies.

NMR and GPC

The fractions of MePEG and alkyl chains on HPGs were estimated fromheteronuclear single quantum coherence (HSQC) NMR experiments recordedon a Bruker Avance 400 MHz (magnetic field strength 9.4 T) NMRspectrometer using deuterated solvents (Cambridge Isotope Laboratories,99.8% D). Molecular weights and polydispersities of the polymers weredetermined by gel permeation chromatography with multi-angle laser lightscattering detection (GPC-MALLS).

FIG. 19A shows a representative 2D HSQC spectrum of HPG-C_(8/10)-MePEG.The surface modification of HPG-C_(8/10)-MePEG withN-(2,3-epoxypropyl)-phthalimide) was confirmed by 2D HSQC experiments inwhich the aromatic phthalimide CH groups were identified (¹H chemicalshifts 7.2-7.8 ppm & ¹³C chemical shifts 125-135 ppm). The success ofcleaving the phthalimide groups by hydrazinolysis to generate free aminegroups was monitored by both 1D NMR and 2D HSQC experiments. FIG. 19Bshows a representative 2D HSQC spectrum of HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎,which shows the success of cleavage of the phthalimide protecting groupto generate primary amine groups (2.60-2.80 ppm & ¹³C chemical shifts 45ppm, FIG. 19B). HSQC NMR data confirmed the structure of dHPGs ashyperbranched polymers with branching architectures evident in thespectra (FIG. 19). The mole fraction of C_(8/10) alkyl chains and MePEGon dHPGs can be calculated from the signal integrals in HSQCexperiments. By comparing the integrals of MePEG methoxy-group and theoctyl/decyl glycidyl ether (O/DGE) methyl group to the integral of theTMP CH₃ group, the fractions of MePEG, and O/DGE (mol/mol) werecalculated for each dHPG polymer (Table 10).

Conductometric Titrations and Fluorescamine Assay

The mole fractions of amine groups derivatized on HPG-C_(8/10)-MePEGpolymers were measured by conductometric titration using HCl and NaOH.Conductometric titrations were done on YSI model 35 conductance meterand 3403 cell with platinum electrode at 25° C. A syringe pump (HarvardInstruments) was used to inject a diluted NaOH solution at a constantflow rate of 0.0102 ml/min. For a typical titration, approximately 10 mgof HPG-C_(8/10)-MePEG-NH₂ was dissolved in distilled water and titratedfirst with 0.05 N HCl followed by back titration with 0.05 N NaOH.Conductance of the solution was measured at every 30 s. Potassiumhydrogen phthalate solution (0.05 N) was used for standardizing sodiumhydroxide solution. Based on conductometric titration and molecularweight measurements, the number of moles of amine groups per HPGmolecule was calculated and the values obtained were in the range of50-119 mol/mol which were consistent with the targeted mole ratios ofNH₂ per mol of HPG-C_(8/10)-MePEG-NH₂ (Table 8 & 10).

TABLE 8 Stoichiometry of reagents used for the synthesis ofHPG-C_(8/10)-MePEG-NH₂ HPG-C_(8/10)-MePEG-NH₂ Mass of reagents (g)Target NH₂ substitution HPG-C_(8/10)- Target NH₂ (mol/mol (moles ofNH₂/mole of HPG) MePEG KH EPP HPG) HPG-C_(8/10)-MePEG-NH₂₍₆₁₎ 4 0.2 0.661 HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎ 4 0.45 1.184 121HPG-C_(8/10)-MePEG-NH₂₍₁₆₁₎ 4 0.6 1.575 161 KH, potassium hydride; EPP,N-(2,3-epoxypropyl)-phthalimide). Molecular weight properties ofHPG-C_(8/10)-MePEG, Mw = 83,000 and Mw/Mn = 1.22

The number of moles of amine groups per HPG molecule may also becalculated using fluorescence and molecular weight measurements. Forexample, a fluorescamine assay for NH₂ may be used (Table 9). HPG-NH₂samples were prepared by measuring >5 mg of HPG-NH₂ into a 2 mL LC/MSglass vial and adding an appropriate amount of deionized H₂O to make aconcentrated stock, then sonicating the mixture until the HPG-NH₂ wasdissolved. The stock solution was diluted to 1 mg/mL with deionized H₂Oper mg of HPG-NH₂

Phenylalanine standard was prepared by measuring approximately 1 mg ofphenylalanine into a aluminum micro weighing dish and transferring to a20 mL glass vial, then adding 5 mL of deionized H₂O per mg ofphenylalanine, and sonicating the mixture until the phenylalanine wasdissolved (0.2 mg/mL phenylalanine stock). 60 μL of 0.2 mg/mLphenylalanine stock was transferred into a glass LC/MS vial. 90 μL ofdeionized H₂O was added, and vortexed to mix the solution (80 ng/mLphenylalanine working standard).

40 μL of 1 mg/mL HPG-NH₂ stock was transferred into a 96-well plate, 10μL of deionized H₂O was added (or 50 μL, 40 μL, 30 μL, 20 μL, 10 μL, or0 μL of 80 ng/mL phenylalanine working standard was added and topped upwith deionized H₂O to 50 μL if necessary); and the sample was pipettedto mix. 12.5 μL of sodium borate buffer was added to the well andpipetted to mix. The pipette tip was rinsed with 0.03% fluorescaminesolution twice to coat the tip and prevent dripping. 12.5 μL of 0.03%fluorescamine solution was added to sample well. The sample was pipettedto mix and then put on a plate shaker, covered, and shaken for 1 min.175 μL of deionized H₂O was added to react excess fluorescamine andpipetted to mix, and briefly placed on the plate shaker. The sample wasanalyzed by a fluorescence plate reader set at an excitation wavelengthof 390 nm, an emission wavelength of 475 nm, and a 5 nm bandwidth forboth excitation and emission wavelengths.

TABLE 9 Mols of amine per mols of HPG measured by fluorescamine assayEPP-A or HPG-MePEG-NH₂ HPG EPP-S KH^(c) or Amine Sample # (g) (mg)NaH^(d) (mg) (mol/HPG mol) 1 16 4720^(a)  540^(c) 10.25 2 2 620^(a) 68^(c) 9.72 3 2 600^(a) 220^(c) 11.24 4 2 636^(a) 560^(c) 7.38 5 2631^(a) 560^(d) 10.42 6 2 604^(b) 250^(c) 13.63 7 2 1200^(b)  504^(c) 378 1 300^(b)  30^(c) 37.56 ^(a)EPP-A (EPP from Atlantic) Purity: 78% byUPLC and 69% by NMR. ^(b)EPP-S (EPP from Sigma-Aldrich) Purity: 91% byUPLC and 95% by NMR.Thermal Analysis

DSC and TGA were used to evaluate the thermal and degradation propertiesof the dHPGs. Thermal analysis was conducted using a TA Instruments DSCQ100 and a TGA Q50. DSC runs were obtained by cycling weighed samples inhermetic sealed aluminum pans through a “heat-cool-heat” cycle at 10°C./min over the temperature range of −90 to 85° C. TGA runs wereconducted primarily in a “stepwise isothermal” mode where each phase ofweight loss in the degradation process was observed under isothermalconditions and the HPGs were heated through to near 100% weight loss at500° C. Table 10 shows the thermal events of HPG-C_(8/10)-MePEG andHPG-C_(8/10)-MePEG-NH₂ samples. HPG-C_(8/10)-MePEG andHPG-C_(8/10)-MePEG-NH₂ samples showed similar DSC/TGA profiles. HPGsexhibited a glass transition temperature between −45 to −58° C. Thepresence of amine groups produced a small increase in the Tg of HPGs.The major degradation event was observed at temperatures above 300° C.,which shows good thermal stability properties of HPGs. Approximately3-5% weight loss occurred at temperatures below 100° C. probably due tosome residual solvents or water in HPGs. A good thermal stability ofHPGs is desirable for pharmaceutical applications to allow for thepotential use of heat sterilization methods.

Particle Sizing and Zeta Potential

Particle size and zeta potential analysis were conducted using a MalvernNanoZS Particle Size analyzer using DTS0012 disposable sizing cuvettesfor each analysis. Samples were filtered with 0.2 μm in-line syringefilter (PALL Acrodisc 13 mm with nylon membrane). Sample acquisitionparameters were: angle at 173° back-scatter with automatic attenuation;number of runs 11 (10 seconds/run); dispersant was water at 25° C.(viscosity 0.8872 cP and RI 1.330); Mark-Houwink parameter A=0.428 andK=7.67e⁻⁰⁵ (cm²/s). HPGs were assumed to have a similar refractive indexas polyethylene glycol (PEG) with a RI=1.460 and absorption 0.01. Thefinal data represented the average of all the runs. HPGs are smallnanoparticles with hydrodynamic radii<10 nm. HPGs form extremely smallnanoparticles of less than 10 nm. Table 10 shows the particle sizes andzeta potential characteristics of HPGs. The surface derivatization ofHPG-C_(8/10)-MePEG with amine groups had no effect on their particlesize, however, a significant effect on zeta potential was observed. Thezeta potential of amine terminated HPG polymers was highly positive atlow pH and changed to slight negative values in basic conditions. ThispH titratable change in surface charge arises fromprotonation/deprotonation of the amine groups. At physiological pH of7.4 some of the amine groups on HPGs are ionized, and therefore,positive zeta potentials were expected (Table 10). However, at pH valuesgreater than 8 essentially all the amine groups are uncharged so theslight negative charge observed at pH 11 was probably due to theelectronegative hydroxyl groups present on these HPGs. Drug loading ofHPGs had no significant effect on their particle sizes and the HPGsremained well dispersed as unimolecular micelles in solution. DTX loadedHPGs nanoparticles were physically stable, no drug precipitation oraggregation observed during one week storage at room temperature.

TABLE 10 Physicochemical characteristics of HPGs derivatized withC_(8/10) alkyl chains and modified with MePEG and amine groups Structureby NMR Titration Zeta Particle Polymers (mol/mol HPG) NH₂ DSC/TGApotential size HPGs MePEG O/DGE (mol/mol) Tg(° C.)¹ Td(° C.)² (mV) (nm)HPG-C_(8/10)-MePEG 4.3 4.7 — −58.9 344 −1.5 7.5 ± 1.0HPG-C_(8/10)-MePEG-NH₂₍₆₁₎ 3.5 2.9 50 −46 325 11 7.7 ± 2.9HPG-C_(8/10)-MePEG-NH_(2(121)*) 8.1 6.3 104 −45.5 327 11.9 9.6 ± 4.5HPG-C_(8/10)-MePEG-NH₂₍₁₆₁₎ 7.9 6.3 119 −46.4 320 13 8.1 ± 2.8 *Thisbatch was selected for drug loading and in vivo studies; ¹Tg, glasstransition taken at midpoint of transition; ²Td, degradation temperaturetaken at maximum weight loss

Example 16: Evaluation of Mucoadhesive Properties

To evaluate the mucoadhesive properties of HPGs, the mucin-particlemethod developed by Thongborisute and Takeuchi was used (Thonghorisute,J.; Takeuchi, H. Int. J. Pharm. 2008, 354, 204-209). This method isbased on changes in particle size due to aggregation of submicron-sizedmucin as a result of interaction between adhesive polymer and mucin.Submicron-sized mucin solution was mixed with equal volumes of HPGs (10%w/v) in 100 mM acetate buffer, vortexed and incubated at 37° C. for 30min. Changes in particle size were monitored by light scatteringmeasurements using 3000 HS Zetasizer (Malvern Instruments, SanBernardino, Calif.). Each test was performed in triplicate and chitosansolution (1% w/v) was used as a positive control. The particle size ofthe mucin increased significantly after incubation with either chitosan(1% w/v) or HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎ (10% w/v) solutions (FIG. 20).HPG-C_(8/10)-MePEG (10% w/v) had no effect on the size of mucinparticles. The increased particle size of the submicron-sized mucin wasdue to aggregated particles of mucin and HPG-C_(8/10)-MePEG-NH₂nanoparticles and was attributed to mucoadhesive forces between mucinand the amine substituted HPGs. Chitosan, a widely known mucoadhesivepolymer was used as a positive control. However, due to its highmolecular weight and low solubility in aqueous solution, a dilutedsolution of chitosan was used (1% w/v). Even this diluted solutionexhibited significant changes in particle size of the mucin afterco-incubation. The mucoadhesiveness of both chitosan andHPG-C_(8/10)-MePEG-NH₂ is believed to be due to electrostaticinteractions between positively charge amine groups and negativelycharged mucin particles but also other contribution such as hydrogenbonding, hydrophobic effects and chain entanglement might have aneffect. However, the lack of mucoadhesiveness of HPG-C_(8/10)-MePEGsuggests that electrostatic attraction appears to be a majorcontribution to the mucoadhesive properties of HPG-C_(8/10)-MePEG-NH₂.

Example 17: Cell Proliferation/Binding and Uptake Studies

Cell Proliferation

KU7-luc cells were plated at 5,000 cells/well in 96-well plates in a 100μl volume of McCoy's Medium supplemented with 10% FBS and allowed toequilibrate for 24 h before freshly prepared solutions ofHPG-C_(8/10)-MePEG and/or HPG-C_(8/10)-MePEG-NH₂ (dissolved in PBS, pH7.4, 0-150 μg/ml) were added. Cells were exposed to the HPG solutionsfor 2 h and cell viability was determined after 72 h using theCellTiter96 AQueous Non-Radioactive Cell Proliferation Assay (Promega,Madison, Wis.) as described previously (Mugabe, C.; Hadaschik, B. A.;Kainthan, R. K.; Brooks, D. E.; So, A. I.; Gleave, M. E.; Burt, H. M.BJU Int. 2009, 103, 978-986).

Rhodamine Labeling of HPGs

HPG-C_(8/10)-MePEG and HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎ polymers werecovalently labeled with tetramethyl-rhodamine-carbonyl-azide (TMRCA) aspreviously reported (Savic, R.; Luo, L.; Eisenberg, A.; Maysinger, D.Science 2003, 300, 615-618). Tetramethylrhodamine-carbonyl-azide (TMRCA)was purchased from Invitrogen Canada Inc. (Burlington, ON). Briefly, 500mg HPGs (HPG-C_(8/10)-MePEG or HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎) weredissolved in 5 ml of anhydrous 1,4-dioxane. An appropriate amount oftetramethylrhodamine-5-carbonyl azide (TMRCA, MW 455.47) was dissolvedin 2 ml anhydrous 1,4-dioxane to give a final concentration of 1 mg/ml.An aliquot of 675 μl of this fluorescent probe, which corresponds toapproximately 20 mol % of HPGs, was added to the HPGs solution andheated at 80° C. in oil bath under nitrogen stream with stirring for 5h. Unreacted probe was removed by dialysis against DMF (MWCO12,000-14,000) until the dialysate was colourless and then dialysedagainst distilled water for 24 h. The fluorescent-labeled polymers(HPGs-TMRCA) were freeze dried and stored at −80° C. in amber vials.

Cell Binding and Uptake

KU7-luc cells were used to assess the binding and uptake of rhodaminelabeled HPGs. Cells were plated at 10,000 cells/well into 96-well platesin 100 μl volume of McCoy's Medium supplemented with 10% PBS and allowedto equilibrate for 24 h. The media was removed and cells were incubatedwith rhodamine labeled HPGs (HPG-C_(8/10)-MePEG-TMRCA orHPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎-TMRCA, 1.56-200 μg/ml) for 2 h. Followingincubation period, cell were washed 3 times with PBS and lysed with 200μl of 0.5% Triton X-100 (pH 8 in PBS) and the amount of cellularfluorescence binding was measured by fluorescence spectroscopy (Synergy4.0) at excitation/emission of 545/578. Standards were prepared fromrhodamine labeled HPGs (0.781-6.25 μg/ml) in Triton X-100 and PBS (pH8). The amount of rhodamine labeled HPGs taken up into cells or surfacebound was expressed as percentage of total amount of polymer added oneach well.

Confocal Fluorescence Analysis of Cell Uptake

KU7-luc cells were grown in 10 cm petri dishes with 1 cm×1 cm coverslips on the bottom of the dish for the cells to grow on, until aconfluence of ˜75% was reached, which corresponded to a cell number of7×10⁴ cells. Cell-containing cover slips were then removed and washed 3times with warmed PBS. Cover slips were then placed cell side up inparafilm lined petri dishes for the duration of the uptake assay.Rhodamine labeled HPG polymers (250 μl of HPG-C_(8/10)-MePEG-TMRCA orHPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎-TMRCA) were added to the cells on coverslips at a concentration of 1 mg/ml. Cells were incubated for 1, 4, 8and 24 h. After each time point, cover slips were washed 4 timesvigorously in PBS. After gently blotting off excess PBS, 250 μl of 3.7%paraformaldehyde at room temperature was used to fix the cells for 10minutes. Cover slips were then washed 3 more times in PBS, submerged inwater, excess liquid was blotted off and were finally mounted cell sidedown on glass slides with Prolong gold with4′,6-diamidino-2-phenylindole (DAPI). Clear nail polish was usedsparingly around the edges of the cover slip to stop drying out thesample. An overnight incubation ensured proper hardening of the samplewhich was then ready for imaging. Microscopy studies were performed onOlympus FV-1000 inverted confocal microscope. The laser wavelengths usedwere 568 nm and 405 nm for imaging of rhodamine and DAPI, respectively.Direct contrast (DIC) was also performed to visualize cell membranes andwas activated with the 405 nm laser as well. Laser power and highvoltage gain was kept relatively constant within each polymer group toallow for consistent comparison. In order to clearly show that labeledpolymer was inside the cell, images were analyzed by fluorescence andDIC.

Cell Proliferation/Binding and Uptake Studies

At low concentrations, HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎-TMRCA nanoparticleswere extensively bound and internalized into KU7-luc, whereas, noevidence of cell binding or uptake was observed forHPG-C_(8/10)-MePEG-TMRCA nanoparticles at concentrations below 12.5μg/ml (FIG. 21A). The strong binding profile of this polymer is probablydue to the electrostatic attraction between the positively chargedHPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎ polymer and the negatively charged cellmembrane of KU7-luc. However, as the concentration of HPGs increased,the cell binding and uptake of HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎-TMRCAnanoparticles reached a saturation point (at 25 μg/ml) while the cellbinding and uptake of HPG-C_(8/10)-MePEG-TMRCA nanoparticles intoKU7-luc was found to be concentration-dependent with linear relationshipobserved at concentrations between 12.5 and 50 μg/ml, followed by lesspronounced binding and uptake at higher polymer concentrations (FIG.21A). To evaluate whether the saturation behavior ofHPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎-TMRCA nanoparticles was due to theircytotoxicity effects, we have evaluated the effect of HPG on theproliferation of the KU7-luc cells. Cells were exposed to empty (nondrug loaded) HPG-C_(8/10)-MePEG and HPG-C_(8/10)-MePEG-NH₂ nanoparticles(0-150 μg/ml) for 2 h and cell viability was determined by MTS assay.Both HPG-C_(8/10)-MePEG and HPG-C_(8/10)-MePEG-NH₂ nanoparticlesexhibited similar proliferation effect and were biocompatible with theKU7-luc cell line at the concentrations tested (FIG. 21B). Therefore,the differences in cell binding and uptake observed forHPG-C_(8/10)-MePEG-TMRCA and HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎-TMRCAnanoparticles were likely not due to their cytotoxicity effect on theKU7-luc cell line.

Confocal Fluorescence Analysis of Internalization of Rhodamine LabeledHPGs

Confocal microscopy was used to monitor whether the nanoparticles wereinternalized by cells or simply bound to the cell membrane of KU7-luc.Both HPG-C_(8/10)-MePEG-TMRCA and HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎-TMRCAnanoparticles were rapidly internalized by KU7-luc cells and completeuptake was attained by 1 h of incubation (data not shown). The presenceof rhodamine labeled HPGs in the cytoplasm was observed by thefluorescence analysis. The presence of HPG-C_(8/10)-MePEG-TMRCA and/orHPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎-TMRCA nanoparticles throughout the cytoplasmwas observed as opposed to being only adhered to or present in cellmembrane. There was no fluorescence from the polymers detected in thenuclear compartment of the KU7-luc cells. The absence of HPGnanoparticles in the nuclear compartment may have been due to theirrelatively high molecular weights (<80 kDa). BothHPG-C_(8/10)-MePEG-TMRCA and HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎-TMRCAnanoparticles have no effect on the viability and prevalence of theKU7-luc cells when compared to the control cells at all time points.Overall, rhodamine labeled HPG nanoparticles were taken up into KU7-luccells by 1 h of incubation and there were no differences in the imagesobtained at 1, 4, 8 or 24 h time points.

Example 18: Loading and Quantification of DTX in HPGs and DTX Releasefrom HPGs

Loading and Quantification of DTX in HPGs

DTX was loaded in HPG-C_(8/10)-MePEG and HPG-C_(8/10)-MePEGNH₂₍₁₂₁₎ by asolvent evaporation method in which the drug and polymer were dissolvedin a common organic solvent and the solvent removed. The resultingpolymer/drug matrix was reconstituted with 10 mM PBS (pH 7.4). Theresulting solutions were generally clear but in cases where whiteparticles were observed, the solutions were centrifuged (18,000 g for 10min) and supernatants were transferred to new vials.

The amounts of DTX incorporated in HPGs were determined by reversedphase HPLC. Drug content analysis was performed using a symmetry C18column (Waters Nova-Pak, Milford, Mass.) with a mobile phase containinga mixture of acetonitrile, water, and methanol (58:37:5, v/v/v) at aflow rate of 1 ml/min. Sample injection volumes were 20 μl and detectionwas performed using UV detection at a wavelength of 232 nm. Total runtime was set to 5 min and DTX retention time was 2.9 min. Up to 5% w/wof drug loading was achieved by this method, which corresponds to about5-6 DTX molecules per HPG molecule. The aqueous solubility of DTX is inthe range of 7 μg/ml (Du, W.; Hong, L.; Yao, T.; Yang, X.; He, Q.; Yang,B.; Hu, Y. Bioorg. Med. Chem. 2007, 15, 6323-6330; Liggins, R. T.;Hunter, W. L.; Burt, H. M. J. Pharm. Sci. 1997, 86, 1458-1463) andincorporation DTX in HPGs resulted in approximately 1,000-fold increasein water solubility of this drug.

DTX Release from HPGs

DTX release from HPG nanoparticles (HPG-C_(8/10)-MePEG andHPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎) was determined by the dialysis method.Briefly, 100 mg of HPG-C_(8/10)-MePEG or HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎were weighed and mixed with 1 mg of DTX in 1 ml acetonitrile solution,spiked with 15 μCi of ³H-DTX and then dried under a nitrogen stream toremove the solvent. The HPG/DTX matrix was hydrated with 2 ml of PBS andtransferred into the dialysis bags and dialysed against 500 ml ofartificial urine (pH 6.5) with shaking at 100 rpm. The pH of thesolution was adjusted to 6.5 using 0.1M HCl. The pH 6.5 was chosenbecause it is the median physiological range for human urine, althoughit may vary over a wide range (pH 4.5-8). (Brooks, T.; Keevil, C. W.Lett. Appl. Microbiol. 1997, 24, 203-206.) At different time points, thevolumes of the dialysis bags were measured and a 10 μl sample was takenfor measurement of the remaining radioactivity in the dialysis bags andthe entire external release media was exchanged with fresh media tomaintain sink conditions. The concentration of ³H-DTX remaining in thedialysis bag at each time point was determined by beta scintillationcounting (Beckman Coulter Canada, Mississagua, ON). The cumulativepercent drug released was calculated by subtracting the amount of drugremaining at each time point from the initial amount of drug at thebeginning of the experiment. The data were expressed as cumulativepercentage drug released as a function of time. The release profiles ofDTX from HPGs were characterized by a continuous controlled release withlittle or no burst phase of release (FIG. 22). Approximately 55% ofinitially encapsulated drug was released within the first 24 h ofincubation. The presence of amine groups on the surface of HPGs had noeffect on drug release (FIG. 22).

Example 19: Evaluation of Intravesical DTX Formulations in an OrthotopicBladder Cancer Model

Tolerability and efficacy of intravesical DTX loaded HPG formulations inmice bearing orthotopic bladder xenografts were evaluated. Theorthotopic mouse model used has been reported (Mugabe, C.; Hadaschik, B.A.; Kainthan, R. K.; Brooks, D. E.; So, A. I.; Gleave, M. E.; Burt, H.M. BJU Int. 2009, 103, 978-986; Hadaschik, B. A.; Black, P. C.; Sea, J.C.; Metwalli, A. R.; Fazli, L.; Dinney, C. P.; Gleave, M. E.; So, A. I.BJU Int 2007, 100, 1377-1384; Hadaschik, B. A.; ter Borg, M. G.;Jackson, J.; Sowery, R. D.; So, A. I.; Burt, H. M.; Gleave, M. E. BJUInt. 2008, 101, 1347-1355). All animal studies were carried out inaccordance with the Canadian Council on Animal Care and the animal careprotocol has been approved by the Animal Care Committee from ourinstitution (The University of British Columbia). In this model,luciferase expressing KU7-luc cancer cells were used. For tumourinoculation, eight-week-old female nude mice (Harlan, Indianapolis,Ind.) were anaesthetized with isoflurane. A superficial purse-stringsuture was placed around the urethral meatus before a lubricated 24GJelco angiocatheter (Medex Medical Ltd., Lancashire, UK) was passedthrough the urethra into the bladder. After a single irrigation of thebladder with 100 μl PBS, two million KU7-luc cells were instilled as asingle cell suspension in 50 μl and the purse-string suture was tieddown for a 2.5 h period of time, during which the mice were keptanaesthetized. After removal of the suture mice were placed in cages andmonitored until they have regained consciousness and voiding in normalmanner. Five days post-tumour inoculation, 26 randomized mice weretreated via intravesical instillation (50 μl and 2 h dwell time)according to the following treatment groups: PBS (control); Taxotere®(0.5 and 1.0 mg/ml, DTX in Tween 80); DTX in HPG-C_(8/10)-MePEG (0.5 and1.0 mg/ml); DTX in HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎ (1.0 mg/ml). Mice weremonitored for several hours on the day of treatment and dailythereafter. Any signs of toxicity were reported, in particular, weightchange in food and water consumption, lethargy, hunched posture, and/orgross manifestations of stress. Any mouse showing signs of pain orillness which did not recover within 24 h was sacrificed. Tumour burdenwas monitored by non-invasive imaging of mice on days 2, 8, 12, and 19with an IVIS 200 Imaging System (Xenogen Corp., Alameda, Calif.).Briefly, mice were injected intraperitoneally with 150 mg/kg luciferin,anaesthetized with isoflurane and imaged in the supine position exactly15 min after luciferin injection. Data were acquired and analyzed usingLiving Image software version 2.50 (Xenogen).

Two days post-tumour inoculation all mice developed bladder tumours,however, 2 mice also developed kidney tumours as demonstrated bybioluminescence imaging (FIG. 23A). Overall, intravesical DTX either thecommercial Taxotere® or HPGs formulations were well tolerated by mice.No major toxicities were observed and all mice survived until the end ofthe study period. However, on day 8 post-tumour inoculation, some micelost about 5% of their body weight, although, they recovered thefollowing week. Body weight loss might have been a result ofintravesical treatment and/or less food and water consumption on thedays following treatment. However, there was no significant difference(p>0.05) in body weight loss between different groups.

Doses of 0.5 and 1.0 mg/ml were selected to establish an appropriatedosing regimen for intravesical DTX in mice bearing bladder cancerxenografts. Mice treated with a single dose of either Taxotere® at 1.0mg/ml, DTX in HPG-C_(8/10)-MePEG at 0.5 mg/ml, HPG-C_(8/10)-MePEG and/orHPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎ at 1 mg/ml strongly inhibited the tumourgrowth. On day 19 post-tumour inoculation, all treatment groups exceptthat of Taxotere® (0.5 mg/ml) showed statistically significant tumourinhibition compared to PBS control (p<0.001, post-hoc Bonferoni analysisafter 2-Way ANOVA). All treatment groups were statisticallysignificantly different compared to the Taxotere® (0.5 mg/ml) group(FIG. 23B, p<0.05, post-hoc Bonferoni analysis after 2-Way ANOVA).

It is believed that mucoadhesive properties of these nanoparticlesincrease the intimacy of contact with the urothelium leading to enhanceddrug permeability and uptake into the bladder wall possibly due to themodulation of tight junctions or desquamation of urothelium. Due totheir very small size (Rh<10 nm), HPGs might diffuse through the mucinglycoproteins and interact directly with the umbrella cells ofurothelium leading to enhanced endocytosis of these nanoparticles intothe bladder wall or tumour tissues.

A second study was conducted to evaluate the effectiveness of a lowerinstillation dose of DTX in HPGs. For this study, mice that had noapparent bladder tumours or with low level of bioluminescence asdetermined by IVIS 200 Imaging System on day 19 were used. In total, 12mice were found to be suitable for a second tumour re-inoculation asdescribed above. Tumour take was about 75% compared to 100% in previousstudies and may be due to an immune response, since the mice werepreviously inoculated with the same cell line; despite use of athymicimmunocompromised mice, these mice still have an inherent local immunesystem characterized by macrophages and natural killer cells. From the 9mice that developed bladder tumours after re-inoculation, 2 micedeveloped even larger (10-100 fold) bladder tumours. On day fivepost-tumour re-inoculation, mice which developed bladder tumours wererandomized in two groups to receive a single 50 μl intravesical DTX (0.2mg/ml) loaded HPG-C_(8/10)-MePEG (n=5) or HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎(n=4). Mice were imaged on days 5, 11 and 19 post-tumour re-inoculation.Intravesical DTX loaded HPG-C_(8/10)-MePEG-NH₂₍₁₂₁₎ inhibited tumourgrowth in mice while DTX loaded HPG-C_(8/10)-MePEG failed to do so atthe same concentration. At day 11 and 19 post-tumour re-inoculation, 3out 4 mice showed no evidence of tumour growth following a singleintravesical treatment with DTX (0.2 mg/ml) loaded HPG-C_(8/10)MePEG-NH₂₍₁₂₁₎ nanoparticles whereas, 4 out of 5 mice treated with DTX(0.2 mg/ml) loaded HPG-C_(8/10)-MePEG nanoparticles showed evidence ofbladder tumour growth and one mouse further developed kidney tumours(FIG. 24). Once again, these formulations were well tolerated by mice nomajor toxicities or body weight loss occurred during these studies.

Example 20: In Vitro Cytotoxicity Studies

Cytotoxic effects of the commercial formulation of Taxotere® and DTXloaded HPG formulations against the KU7-luc cell line, and bothlow-grade (RT4, MGHU3) and high-grade (UMUC3) human urothelial carcinomacell lines were evaluated.

Taxotere® (DTX in Tween 80) was purchased from Sanofi-Aventis CanadaInc. (Laval, Quebec). The human bladder cancer cell lines RT4 and UMUC3were purchased from the American Type Culture Collection. Cells weremaintained in McCoy's medium (Invitrogen, Burlington, ON) containing 10%heat-inactivated fetal bovine serum and kept at 37° C. in a humidified5% CO₂ atmosphere. MGHU3 cells were obtained as a generous gift from Dr.Y. Fradet (L'Hotel-Dieu de Quebec, Quebec, Canada) and maintained in MEMsupplemented with 10% fetal bovine serum and 2 mM L-glutamine(Invitrogen). KU7 was kindly provided by Dr. C. Dinney (M D AndersonCancer Center, Houston, Tex., USA) and maintained in DMEM containing 5%fetal bovine serum. For visualization purposes, KU7 cells were infectedwith a lentivirus containing the firefly luciferase gene by Dr. GraigLogsdon (M. D. Anderson Cancer Center, Houston, Tex., USA), and thesesubclones were named KU7-luc as described previously (Hadaschik B A,Black P C, Sea J C, et al. BJU Int 2007; 100: 1377-84).

Cells were plated at 5,000 cells/well in 96-well plates in a 100 μlvolume of McCoy's Medium supplemented with 10% PBS and allowed toequilibrate for 24 h before freshly prepared solutions of Taxotere®, orDTX in HPG-C_(8/10)-MePEG and/or HPG-C_(8/10)-MePEG-NH₂ (dissolved inPBS, pH 7.4) were added. Cells were exposed to the drug formulations for2 h, to simulate the current clinical standard for instillation therapy,and cell viability was determined after 72 h using the CellTiter96AQueous Non-Radioactive Cell Proliferation (MTS) Assay (Promega,Madison, Wis.) as previously reported (Mugabe C, Hadaschik B A, KainthanR K, et al. BJU Int 2009; 103: 978-86). Each experiment was repeatedthree times and MTS values fell within a linear absorbance range for allcell lines.

All DTX formulations resulted in concentration-dependent inhibition ofproliferation in all cell lines tested. The more aggressive and fastgrowing KU7-luc cell line was the most sensitive to DTX formulations DTXloaded HPG-C_(8/10)-MePEG or HPG-C_(8/10)-MePEG-NH₂ were found to be ascytotoxic as the commercial formulation of Taxotere® (FIG. 25). The IC₅₀of DTX formulations were in the low nanomolar range (4-12 nM) for allcell lines tested. Control HPGs nanoparticles (no drug) showed nocytotoxicity across the tested concentration range (15-1,500 nM, datanot shown). Loading of DTX in HPGs had no effect on its cytotoxicity.

Example 21: In Vivo Studies

Efficacy of Intravesical DTX in Orthotopic Murine Model of BladderCancer

In vivo studies were done in a total of 42 nude mice to evaluate theefficacy of a single intravesical treatment with Taxotere® (0.2 mg/ml)and DTX (0.2 mg/ml) loaded HPG-C_(8/10)-MePEG and/orHPG-C_(8/10)-MePEG-NH₂. The orthotopic mouse model used has beenreported (Hadaschik B A, Black P C, Sea J C, et al. BJU Int 2007; 100:1377-84; Mugabe C, Hadaschik B A, Kainthan R K, et al. BJU Int 2009;103: 978-86; Hadaschik B A, ter Borg M G, Jackson J, et al. BJU Int2008; 101: 1347-55; Hadaschik B A, Adomat H, Fazli L, et al. Clin CancerRes 2008; 14: 1510-8; Hadaschik B A, Zhang K, So A I, et al. Cancer Res2008; 68: 4506-10). Animal studies were carried out in accordance withthe Canadian Council on Animal Care. Eleven-week-old female nude mice(Harlan, Indianapolis, Ind.) were anaesthetized with isoflurane. Asuperficial 6-0 polypropylene purse-string suture was placed around theurethral meatus before a lubricated 24 G Jelco angiocatheter (MedexMedical Ltd., Lancashire, UK) was passed through the urethra into thebladder. After a single irrigation of the bladder with PBS, two millionKU7-luc cells were instilled as a single cell suspension in 50 μl andthe purse-string suture was tied down for 2.5 h. To quantify in vivotumor burden, animals were imaged in supine position 15 min afterintraperitoneal injection of 150 mg/kg luciferin on days 4, 11, 18, and25 with an IVIS200 Imaging System (Xenogen/Caliper Life Sciences,Hopkinton, Mass.). Data were acquired and analyzed using Living Imagesoftware (Xenogen). On day five post-tumor inoculation, mice wererandomized to receive a single 50 μl intravesical treatment with PBS(control); Taxotere® (0.2 mg/ml); DTX (0.2 mg/ml) loadedHPG-C_(8/10)-MePEG; and DTX (0.2 mg/ml) loaded HPG-C_(8/10)-MePEG-NH₂.Levels of bioluminescence were equivalent among the groups; however, astumors varied between individual mice, for statistical analyses, tumorbioluminescence after treatment was normalized against the initial fluxon day four in each mouse. Necropsy was performed on day 25 after tumorinoculation. The whole bladders were removed, fixed in 10% bufferedformalin and embedded in paraffin. 5 μm sections were prepared andstained with H&E using standard techniques. All slides were reviewed andscanned on a BLISS microscope imaging workstation (Bacus LaboratoriesInc., Lombard, Ill.).

After intravesical inoculation of KU7-luc cancer cells, all micedeveloped bladder tumors. However, one mouse in DTX loadedHPG-C_(8/10)-MePEG-NH₂ group died unexpectedly on day fourpost-treatment. Overall, intravesical DTX administered as either thecommercial Taxotere® or the HPGs formulations were well tolerated bymice and no major toxicities were observed.

Compared with control mice, DTX loaded HPGs inhibited tumor growth.However, DTX loaded HPG-C_(8/10)-MePEG-NH₂ was the most effectiveformulation to inhibit tumor growth in KU7-luc orthotopic bladder cancerxenografts and reached statistical significance compared to either thePBS control or Taxotere® groups (FIG. 26, P<0.01, post-hoc Bonferonianalysis after 2-Way ANOVA). At the end of the study, a singleintravesical instillation of DTX loaded HPG-C_(8/10)-MePEG-NH₂nanoparticles inhibited tumor growth by 88% compared to the PBS controlgroups. DTX loaded HPG-C_(8/10)-MePEG nanoparticles exhibited a 54%tumor inhibition in this treatment arm. This increase in efficacy likelyresulted from enhanced drug uptake in bladder and tumor tissues of micetreated with DTX loaded HPG-C_(8/10)-MePEG-NH₂ nanoparticles.

The commercial formulation of Taxotere® failed to inhibit tumor growthin this orthotopic xenograft model. Representative bioluminescenceimages of mice over time in each treatment group are shown in FIG. 26.Histological examination of bladder tissues show that KU7-luc tumorsexhibited an aggressive growth pattern and frequent multifocality, butafter 25 days post-tumor inoculation, they were generally confined tothe lamina propria and correlated with high-grade T1 stage disease (FIG.27). Although DTX (0.2 mg/ml) did not cause any remarkable histologicalchange in KU7-luc xenograft compared with the PBS treatment, DTX (0.2mg/ml) loaded HPG-C_(8/10)-MePEG and/or HPG-C_(8/10)-MePEG-NH₂ inhibitedtumor growth. Tumors treated by DTX loaded in HPG-C_(8/10)-MePEG-NH₂decreased significantly in size, with heterogeneous cellular size,nuclear shape and infiltrating inflammatory cells.

It was also determined that the technique used for loading thebiologically active moiety into a dHPG is capable of producing aformulation that can be assayed as having ±20% of the target amount ofdrug in the delivery system, as determined by HPLC (see Table 11).

TABLE 11 Concentration of DTX formulations in samples analyzedpost-treatment by HPLC Theoretical Actual Concentration of Concentrationof Formulation Docetaxel (mg/mL) Docetaxel (mg/mL) Taxotere ™ 0.5 0.531.0 0.81 HPG-C_(8/10)-MePEG-NH₂ 0.5 0.48 1.0 0.94 1.0 0.86

A comparison has been made between formulations incorporating paclitaxel(PTX) and those incorporating DTX, as shown in FIG. 28. For both drugs,incorporating them into a dHPG, such as HPG-C_(8/10)-MePEG, results inreduced tumor luminescence, with a dHPG incorporating DTX being moreeffective than a dHPG incorporating paclitaxel.

A similar experiment has been repeated using two time points and usinghealthy animals. These data, shown in FIG. 29 demonstrate that inhealthy mice, more docetaxel is retained in the bladder over time usingHPG-C_(8/10)-MePEG-NH₂ than when using either HPG-C_(8/10)-MePEG orTaxotere™ to deliver the same amount of the drug. Results aresemi-quantitative for the HPG-MePEG-NH₂ due to exceeding the assaysupper limit of quantitation. The results for the Taxotere™ andHPG-C_(8/10)-MePEG group are quantitative. Mice were dosed each with 50μg of drug in a 50 μL volume and 50 mg of dHPG was used (n=3 per group).

Example 22: Drug Uptake Studies

To evaluate the bladder tissue and serum uptake following intravesicalDTX formulations, mice with orthotopic bladder tumors were instilledwith either Taxotere® (0.2 mg/ml, n=3) or DTX (0.2 mg/ml) loadedHPG-C_(8/10)-MePEG (n=4) and/or HPG-C_(8/10)-MePEG-NH₂ (n=4). The amountof DTX in urine, bladder tissue, and serum were measured two hourspost-instillation. Drug uptake studies were conducted infifteen-week-old female nude mice with established KU7-luc tumors (33days post-tumor inoculation). Tail blood samples were taken at 0, 30,and 60 min post intravesical instillation. During this period mice werestill anaesthetized with isoflurane. After 2 h, all mice were euthanizedusing CO₂ asphyxiation and additional blood was removed by cardiacpuncture. Blood samples were centrifuged in micro-haematocrit tubes(Fisher Scientific, Pittsburg, Pa.) or serum-separator tubes (BectonDickinson) and the serum was snap-frozen in liquid nitrogen. Urine andbladder of each mouse were also harvested and before freezing, thebladders were cut, opened to expose the lumen and were vigorously washedin five sequential 10 ml PBS washes. All samples were stored at −80° C.The UPLC-MS/MS system used for analysis consisted of an integratedWaters Acquity UPLC separation system (Acquity BEH C18, 1.7 μm, 2.1×50mm column) coupled to a mass spectometry analysis using Waters TQD massspectrometer. The system was operated at an electrospray ion sourceblock temperature of 150° C., a desolvation temperature of 350° C., acone voltage of 14 V, a capillary voltage of 0.70 kV, extractor voltageof 3 kV, RF voltage of 0.1 kV, a cone gas flow at 25 l/h, a desolvationgas flow at 600 l/h and a collision gas flow at 0.2 ml/min. Themolecules undergo electron spray ionization in the positive ion mode.DTX was quantified in multiple reaction monitoring with the transitionof m/z 808.5→527.2, as previously established (Mugabe C, Liggins R T,Guan D, et al. Int J Pharm 2011; 404: 238-49). DTX was extracted fromthe mouse serum by solvent/solvent extraction method. 50 μl aliquots ofthe mouse plasma and standards were mixed with 150 μl of 0.1% formicacid in acetonitrile in a 96-well plate and vortexed for 1 min at roomtemperature. The samples were centrifuged at 5,500 rpm (Allegra™ 25 Rcentrifuge, Beckman-Coulter) for 10 min at 4° C. Then 100 μl of thesupernatant was mixed with 50 μl of distilled water, mixed and vortexedfor 30 s. Bladder tissues were weighed and homogenized in 0.1% formicacid/methanol using zirconia beads (Biospec Products) and mini-beadbeater equipped with microvial holder (Biospec Products) for 60 s. Thesamples were centrifuged at 14,000 rpm (Allegra™ 25 R centrifuge,Beckman-Coulter) for 2 min at 4° C. 150 μl of 0.1% trifluoroacetic acidin methanol was added to the samples, mixed and vortexed at 14,000 rpm(Allegra™ 25 R centrifuge, Beckman-Coulter) for 15 min at 4° C. Allsample analysis was performed using UPLCMS/MS. The limit ofquantification for DTX was 10 ng/ml with a recovery of 97% from spikedcontrol samples. Within run precision (% RSD) was less than 15% in allcases.

Mice instilled with Taxotere® had no detectable DTX in serum at all timepoints. DTX loaded HPG-C_(8/10)-MePEG-NH₂ exhibited the highest serumlevels at the 2 h time point (150.87±34.98 vs 23.97±16.71 ng/ml, P<0.01,2-way ANOVA, Bonferroni post-test). However, serum concentrations of DTXwere several orders of magnitude lower than the concentrations in urineand bladder tissue (Table 12). DTX loaded HPG-C_(8/10)-MePEG-NH₂resulted in significantly higher amounts in bladder tissue accumulationcompared to Taxotere® or DTX loaded HPG-C_(8/10)-MePEG (P<0.001, 1-wayANOVA, Bonferroni's multiple comparison test). There was no significantdifference (P>0.05, 1-way ANOVA) in bladder tissue accumulation betweenTaxotere® and DTX loaded HPG-C_(8/10)-MePEG treatment groups. The finalurine concentrations were about 5-7-fold lower than the initial dosingsolution. This was due to the urine dilution during the 2 h period ofintravesical instillation. However, there was no significant difference(P>0.05, 1-way ANOVA) in the final urine concentrations of DTX betweendifferent treatment groups. No local or systemic toxicity was observedin either group.

TABLE 12 Drug uptake of intravesical DTX formulations in orthotopicxenografts DTX formulations ¹C_(urine) ²C_(bladder) ³C_(serum)(ng/ml)(No. of mice) (μg/ml) (μg/g) 0.5 h 1 h 2 h Taxotere ® (3) 31.4 ±  1.24 ±BLOQ BLOQ BLOQ 15.5 0.54 DTX/HPG-C_(8/10)- 53.8 ±  1.09 ± 55.87 27.88 23.97 ± MePEG (4) 8.1 0.70 16.71 DTX/HPG-C_(8/10)- 27.6 ± 13.07 ± 81.47± 88.21 ± 150.87 ± MePEG-NH₂ (4) 4.0 4.32 23.76 39.42 34.98 ¹Finalconcentration of DTX in mouse urine after 2 h of intravesicalinstillation measured by HPLC ²Concentration of DTX in mouse bladdertissue following a 2 h intravesical instillation measured by LC/MS/MS³Concentration of DTX in mouse serum taken at 0.5, 1, and 2 hpost-intravesical instillation measured by LC/MS/MS BLOQ, below thelimit of quantification (lowest limit of quantification was 10 ng/ml)Data shown are the mean ± SD

Example 23: Assessing Tumor Microenvironment and Uptake of RhodamineLabeled HPGs

Bladder tumor microenvironment and distribution of rhodamine labeledHPGs into tumor tissue was assessed.

Rhodamine Labeling of HPGs

HPG-C_(8/10)-MePEG and HPG-C_(8/10)-MePEG-NH₂ polymers were covalentlylabeled with tetramethyl-rhodamine-carbonyl-azide (TMRCA) as previouslyreported (Savic R, Luo L, Eisenberg A, Maysinger D. Science 2003; 300:615-8; Mugabe C, Liggins R T, Guan D, et al. Int J Pharm 2011; 404:238-49). Fifteen-week-old female nude mice with orthotopic bladdertumors (33 days post-tumor inoculation) were anaesthetized withisoflurane. A superficial 6/0 polypropylene purse-string suture wasplaced around the urethral meatus and the bladder was emptied by manualcompression. A lubricated 24-gauge telco angiocatheter was passedthrough the urethra into the bladder and then 50 μl of either PBS, freerhodamine (TMRCA), HPG-C_(8/10)-MePEG-TMRCA, and/orHPG-C_(8/10)-MePEG-NH₂-TMRCA was instilled and the purse-string suture,was tied down for a 2-h period, during which the mice were keptanaesthetized. After the 2-h period the purse-string suture was removed,the bladder was emptied by manual compression and washed twice with 150μl of PBS (pH 6.0). The mice were euthanized and the bladders wereexcised and frozen on an aluminum block, then embedded in OCT forcryosectioning. 10 μm cryosections were cut at distances of 1, 2, and 3mm from the bladder edge. Sections were dried at room temperature andimaged for rhodamine fluorescence using 10× objective (0.75 μm/pixelresolution). Slides were fixed in 1:1 acetone:methanol solution for 10min and stained using a custom capillary-action staining apparatus forCD31 (1:50 hamster anti-CD31 with an anti-hamster Alexa 647 secondary)and Hoechst 33342 (nuclear dye). Following fluorescent imaging of CD31and Hoechst 33342, sections were counterstained lightly withhematoxylin, mounted & imaged in bright field.

Image analyses: images were reduced to 1.5 μm/pixel resolution toimprove manageability in Image J software. With user-suppliedalgorithms, image stacks were then created, aligned and cropped to tumortissue boundaries with artifacts removed; necrosis was further croppedbased on the hematoxylin image. The bladder lumen was artificiallytraced along the tumor tissue boundary on Hoechst 33342 images.User-supplied analysis macros were run to generate the following typesof data: a) threshold: was manually determined to include positive stainbut that does not pick up background outside of necrosis areas; themacro determines the number of positive pixels meeting or exceeding thisthreshold and was reported as an average for the whole tumor section. b)intensity: was reported as the average intensity of staining for a wholetumor section, or the average intensity of pixels sorted based on theirdistance from a secondary stain (ie: CD31) or artificially tracedboundary (bladder lumen). Calculations to determine averages±standarderror were performed and graphic displays created using Microsoft Excel;non-parametric analysis of variance (Kruskal-wallis tests) statisticalanalyses were performed using Prism v5 for Macs software.

Bladder tumor microenvironment and distribution of rhodamine labeledHPGs into tumor tissue was assessed. Bladder tumor tissues were hillyvascularised with an average distance of 40-60 μm to the nearest bloodvessel (FIG. 30A). No significant difference was seen between differentgroups (P=0.8). The amount of fluorescence inside whole bladder tumorswas measured. Rhodamine labeled HPG-C_(8/10)-MePEG-NH₂(HPG-C_(8/10)-MePEG-NH₂-TMRCA) exhibited the highest tumor uptakecompared to the other groups (P=0.037). There was no significantdifference (P>0.05) in tumor uptake of the bladders instilled with freerhodamine (TMRCA) and rhodamine labeled HPG-C_(8/10)-MePEG (FIG. 30B).The depth profile of rhodamine uptake into the tumor tissues wasassessed as a function of distance from the bladder lumen.HPG-C_(8/10)-MePEG-NH₂-TMRCA nanoparticles demonstrated enhanced tumoruptake at all distances from lumen, showing a 5-6-fold increase overHPG-C_(8/10)-MePEG-TMRCA nanoparticles (FIG. 30C).

Example 24: Synthesis and Characterization of HPG-C_(8/10)-MePEG andHPG-C_(8/10)-MePEG-COOH

The polymerization of O/DGE core modified HPGs was carried out accordingto protocols described in our previous report (Kainthan, R. K.; Brooks,D. E. Bioconjugate Chem. 2008, 19, 2231-2238). The functionalization ofC_(8/10) core-modified HPGs with carboxylic acid groups was carried outaccording to protocols reported earlier (Haxton, K. J.; Burt, H. M.Dalton Trans. 2008, 5872-5875). For a typical reaction, 5.0 g of theHPG-C_(8/10)-OH or HPG-C_(8/10)-MePEG_(6.5) was dissolved in 100 mL ofpyridine, and the solution was kept under a nitrogen atmosphere,followed by the addition of dimethylaminopyridine and succinicanhydride, which were adjusted according to the target amount ofcarboxylic acid groups on HPGs. For the synthesis of HPG with thehighest amount of COOH groups, all available free hydroxyl groups weretargeted for modification to carboxylates; therefore, an excess amountof dimethylaminopyridine (0.075 g, 0.61 mmol) and succinic anhydride(4.5 g, 45 mmol) were added to the reaction mixture. Through calculationof the theoretical moles of free hydroxyl groups, it was determined thatthere were 348 mols of free hydroxyl groups per mole of HPG and, thus,theoretically the same number of carboxyl groups per mole of HPG.Therefore, the resulting HPG was denoted asHPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈. The use of lower amounts ofdimethylaminopyridine (0.015 g, 0.12 mmol) and of succinic anhydride(0.9 g, 9 mmol) produced HPGs in which not all the free hydroxyls weretargeted for modification. The theoretical number of carboxylate groupsadded to the HPG was determined through the calculation of the number ofmoles succinic anhydride added to the reaction mixture. Therefore, thislow carboxylate containing HPG was denoted asHPGC_(8/10)-MePEG_(6.5)-COOH₁₁₃. After addition of thedimethylaminopyridine and succinic anhydride, the solution was stirredusing a magnetic stir bar overnight at room temperature. Deionized water(100 mL) was added to the flask and the mixture was kept stirring for 30min. Solvents were removed by rotary evaporation with the periodicaddition of water to enable better evaporation of pyridine by azeotropicdistillation. The final products were dissolved in methanol and dialyzedagainst a mixture of 80:20 methanol/deionized water for 3 days usingcellulose acetate dialysis tubing (MWCO 10000 g/mol, SpectrumLaboratories Inc., Rancho Domunguez, Calif.). The dialysis medium waschanged every 8 h, each time with a lower methanol concentration untilduring the final three stages, the dialysis medium was 100% water.Polymers were obtained by freeze-drying.

¹³C NMR of HPG-C_(8/10)-MePEG-COOH (400 MHz, methanol-d₄) δ_(C): 0(tetramethylsilane, internal reference), 14.73 (CH₃, alkyl on O/DGE),23.92-33.24 (C(O)CH₂CH₂COOH), 48.51-49.86 (solvent, methanol-d₄), 59.29(CH₃O-MePEG), 64.19-65.36 (—CH₂OH, unreacted primary alcohol groups inpolymer), 69.98-73.74 (—CH₂—O—, —CH—O in polymer), 78.93-80.14 (CH inpolymer), 173.84-174.16 (C(O)CH₂CH₂COOH), 175.92 (C(O)CH₂CH₂COOH).

In the preparation of all functionalized HPGs, target amounts of MePEGand COOH groups were added to reaction mixtures. The target amounts ofMePEG and COOH of various functionalized HPGs are summarized in Table13. The reaction yields for the high and low carboxylate functionalizedHPGs were 84 and 74%, respectively. HPG polymers are described by thefollowing nomenclature: HPG-C_(8/10)-MePEGA-COOHB wherein HPG-C_(8/10)represents the alkyl substituted HPG, A is the target content of MePEGconjugated to the polymer, based on the stoichiometry of reagents (molesof MePEG/mol of TMP initiator), and B is the expected molar content ofCOOH per mole of HPG polymer, based on the calculated molecular weightof the polymer from GPC data.

TABLE 13 Properties of a Series of Surface-Modified C_(8/10) AlkylDerivatized Hyperbranched Polyglycerols titration data^(b) molecularweight^(c) COOH particle polymer (mol/mol Mw PDI size^(d)composition^(a) HPG) (g/mol) (Mw/Mn) (nm) HPG-C_(8/10)-OH N/A N/D N/D9.2 ± 3.5 HPG-C_(8/10)-MePEG_(6.5) N/A 7.6 × 10⁴ 1.2 8.7 ± 3.8HPG-C_(8/10)-COOH N/D N/D N/D 5.3 ± 1.8 HPG-C_(8/10)- 318 1.3 × 10⁵ 1.45.9 ± 2.1 MePEG_(6.5)-COOH₃₄₈ HPG-C_(8/10)- 87 9.1 × 10⁴ 1.3 7.4 ± 3.0MePEG_(6.5)-COOH₁₁₃ ^(a)Nomenclature is designated as follows.HPG-C_(8/10)-OH is the “base polymer” and all others were surfacemodified with MePEG and COOH, expressed as the theoretical number ofmoles of surface group added in the reaction per mole of HPG. ^(b)Molesof COOH groups per mole of HPG as determined by pH titration. ^(c)Weightaverage molecular weight and polydispersity index determined by GPC.Number average molecular weight is calculated by Mw/PDL ^(d)Particlesize (diameter), as determined by dynamic light scattering.NMR Analysis

After purification, all of the HPGs were characterized by NMR analysis.NMR spectra of HPG polymers were acquired using a 400 MHz Bruker AvanceII+ spectrometer (Bruker Corporation, Milton, ON). Polymers weredissolved in DMSO-d₆ or methanol-d₄ (Cambridge Isotope Laboratories,Andover, Mass.). One-dimensional proton and carbon spectra wereobtained, as well as two-dimensional, multiplicity-edited heteronuclearsingle quantum coherence (HSQC), heteronuclear multiple-bond correlation(HMBC), and HSQC-TOCSY (total correlation spectroscopy) NMR experiments.Chemical shifts were referenced to the residual solvent peak.Two-dimensional spectra were analyzed using Sparky (T. D. Goddard and D.G. Kneller, Sparky 3, University of California, San Francisco). The molefractions of COOH on HPGs were estimated from HSQC data as follows: Foreach of the modifications, the peak corresponding to the four methyleneprotons was integrated and its integral corrected for the number ofprotons. This value was divided by the integral of the TMP methyl group(corrected for proton multiplicity) to yield the mole fraction of COOH.

From FIG. 31 it can be seen that all of the peaks of functionalizedHPG-C_(8/10)-MePEG_(6.5)-COOH polymers were assigned to the structuralcomponents of the HPGs and were consistent with previous reports(Kainthan, R. K.; Mugabe, C.; Burt, H. M.; Brooks, D. E.Biomacromolecules 2008, 9, 886-895; Kainthan, R. K.; Janzen, J.;Kizhakkedathu, J. N.; Devine, D. V.; Brooks, D. E. Biomaterials 2008,29, 1693-1704; Haxton, K. J.; Burt, H. M. Dalton Trans. 2008,5872-5875). HSQC, HMBC, and HSQC-TOCSY were used to estimate thefractions of the substituents, C_(8/10) alkyl chain, MePEG, and COOH onHPGs using integrated peak volumes.

Degree of Branching and Degree of Polymerization

Hyperbranched polymers are typically characterized by the degree ofbranching (DB) and degree of polymerization (DPn) using the followingequations (Holter, D.; Burgath, A.; Frey, H. Acta Polym. 1997, 48,30-35):

${DB} = \frac{2\; D}{{2\; D} + L_{13} + L_{14}}$where DB is the degree of branching, D, L₁₃, and L₁₄ represent thefractions of dendritic, linear 1-3, and linear 1-4 units, respectively.The structures of the dendritic and linear repeat units of glycidol thatare present in the hyperbranched structure are summarized in FIG. 32.Furthermore, the degree of polymerization (DPn) for these polymers iscalculated as follows (Sunder, A.; Hanselmann, R.; Frey, H.; Mulhaupt,R. Macromolecules 1999, 32, 4240-4246):

${DP}_{n} = {\frac{T + L_{13} + L_{14} + D}{T - D} \cdot f_{c}}$where D, L₁₃, and L₁₄ are defined as above, T represents the fraction ofterminal units, and fc is the functionalization of the core molecule(which is 3 for TMP). D is given by the sum of primary and secondaryunits, Dp and Ds (see FIG. 32), and L₁₃, L₁₄, and T are defined in ananalogous manner.

When a combination of 2D HMBC and HSQC-TOCSY experiments is used, anumber of peaks corresponding to primary and secondary L₁₃, L₁₄, T, andD units were assigned for an unmodified HPG polymer (which wassynthesized as a reference material, containing no C_(8/10) alkylcomponent and no MePEG addition or carboxyl modification, data notshown), and peak volumes from a multiplicity-edited HSQC were used tocalculate DB and DPn. The results obtained for our unmodified HPG wereDB) 0.51 and DPn) 14.83, and the relative abundances for structuralunits are 39% for linear units, 20% for dendritic units, and 41% forterminal units. These values are in good agreement with literaturevalues (Sunder, A.; Hanselmann, R.; Frey, H.; Mulhaupt, R.Macromolecules 1999, 32, 4240-4246; Holter, D.; Burgath, A.; Frey, H.Acta Polym. 1997, 48, 30-35). When comparing the HSQC spectrum of HPGmodified with C_(8/10) alkyl chains (HPG-C_(8/10)-OH), to the HSQCspectrum of unmodified HPG, two new peaks are visible in the spectralregion of the polymer core of the former (FIG. 33). One peak wasassigned to the R-methylene group of the aliphatic chain, whereas thesecond peak could not be assigned unambiguously. Based on chemicalshifts, this peak may correspond to a T unit with one alkyl chainattached to the secondary hydroxyl group; however, this speculationcould not be confirmed. A similar situation was observed forHPG-MePEG_(6.5). The peak from the R-methylene group of the MePEG couldbe assigned, but the additional, unknown peak could not be assignedunambiguously. Similar to HPG-C_(8/10)-OH, the chemical shifts of thenew peak are similar to an L₁₄-like unit. In summary, DB and DPn couldnot be calculated from NMR data due to lack of unambiguous signalassignment. NMR data allows for a straightforward characterization offree hydroxyl groups through observation of linear or terminal units andconfirmation that all expected branching patterns and modifications(alkyl, MePEG, and carboxyl) are present. FIG. 33 illustrates thevarious assigned peaks in the NMR spectra, showing the presence of theexpected branching pattern, and of MePEG, alkyl chains, and COOH groups.

Mole Fractions of COOH

For all HPG polymers modified with COOH, the mole fractions of COOH wereestimated from HSQC NMR spectra. By this method, the number of COOH inthe HPG polymer is not an absolute number, because it is expressed asrelative to the TMP methyl groups present in the sample. Each HPGmolecule is assumed to contain only one TMP; however, the amount of TMPper mole of HPG in the various batches of polymer has not beenindependently quantified. Therefore, these numbers serve as aqualitative indicator of how many hydroxyl groups were capped with COOH.Furthermore, because the HPG-C_(8/10)-MePEG_(6.5)-COOH polymers wereboth synthesized from the same batch of HPGC_(8/10)-MePEG_(6.5), the TMPcontent is expected to be identical and the NMR spectra can be comparedto determine the relative amount of COOH in the twoHPG-C_(8/10)-MePEG_(6.5)-COOH polymers. The molar ratios indicate thatthere is a 2.8-fold higher COOH content inHPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ compared with theHPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃. This is in good agreement with the3.1-fold ratio of target COOH content in the two polymers. ForHPG-C_(8/10)-COOH and the high-carboxyl densityHPGC_(8/10)-MePEG_(6.5)-COOH₃₄₈ polymers, no peaks corresponding tolinear or terminal groups were observed, indicating that no hydroxylgroups are present in this polymer (see FIG. 34 for a representative NMRspectrum). For the lower density COOH polymer,HPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃, peaks of linear and terminal groupswere observed in addition to the new peaks, indicating only a partialsaturation of hydroxyl groups with carboxylic acids (data not shown).

FT-IR

FT-IR spectra for HPGs were obtained using a Perkin-Elmer FTIRspectrometer (Perkin-Elmer, Woodbridge, ON) with a universal ATRsampling accessory. The scanning range was 4000-650 cm⁻¹ with aresolution of 4 cm⁻¹.

FT-IR spectra of HPG-C_(8/10)-MePEG_(6.5),HPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ and HPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃are shown in FIG. 35. The peak at 2800-3000 cm⁻¹ is consistent with C—Hvibrations and occurs in all HPGs. The peaks at 1680-1780 cm⁻¹ arosefrom CdO bands, indicating the presence of COOH groups in theHPG-C_(8/10)-MePEG-COOH polymers. The peaks at 1200-1400 and 1000-1180cm⁻¹ arise from a C—H bend and C—O vibration, respectively, andtherefore, they can be found in all of these polymers. By comparing theFT-IR spectra of the HPG-C_(8/10)-MePEG_(6.5), theHPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃, and theHPG-C_(8/10)-Me-PEG_(6.5)-COOH₃₄₈, it can be seen that the OH peak(3300-3500 cm⁻¹) decreases and the CdO peak (1680-1780 cm⁻¹) in theHPG-C_(8/10)-MePEG-COOH appears, indicating the OH groups have beenconsumed and converted to COOH. The spectrum of theHPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ showed the near elimination of the OHpeak, indicating that the OH groups were largely consumed and convertedto COOH, whereas the OH peak for HPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ wasdecreased but still evident, in agreement with the NMR results.Furthermore, the latter HPG also shows a smaller CdO peak, indicating alower mole ratio of COOH compared to HPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈.The FT-IR data showed good evidence to support the changes in thefunctionalization of the HPGs and also confirmed that unreacted reagentswere removed by the purification procedures.

Molecular Weight

Weight average molecular weights (Mw) and polydispersities (PDI) of theHPGs were determined by gel permeation chromatography (GPC) equippedwith a DAWN-EOS multiangle laser light scattering (MALLS) detector(GPC-MALLS) and Optilab RI detector (Wyatt Technology Inc., SantaBarbara, Calif.). Aqueous 0.1 N sodium nitrate solution was used as themobile phase at a flow rate of 0.8 mL/min. The details have beendescribed in a previous report (Kainthan, R. K.; Brooks, D. E.Bioconjugate Chem. 2008, 19, 2231-2238; Kumar, K. R.; Kizhakkedathu, J.N.; Brooks, D. E. Macromol. Chem. Phys. 2004, 205, 567-573). The dn/dcvalues for various HPGs were determined to be 0.146, 0.165, and 0.138for HPGC_(8/10)-MePEG_(6.5), HPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈, andHPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃, respectively, in aqueous 0.1 N NaNO₃solutions and were used for the calculation of molecular weight ofpolymers. The data were processed using Astra software provided by WyattTechnology Corp. Number average molecular weights of the polymers werecalculated by dividing Mw by PDI.

The molecular weights and polydispersities of these HPGs are shown inTable 13. The functionalized HPGs (HPG-C_(8/10)-MePEG_(6.5)-COOH) showedincreases in molecular weight compared to HPG-C_(8/10)-MePEG_(6.5).Furthermore, it was found that after the surface functionalization, thepolydispersities of the polymers were not altered greatly, indicating arelatively uniform surface modification. Molecular weight values weresimilar to those of previously reported HPG-C_(8/10)-MePEG (Kainthan, R.K.; Mugabe, C.; Burt, H. M.; Brooks, D. E. Biomacromolecules 2008, 9,886-895; Kainthan, R. K.; Janzen, J.; Kizhakkedathu, J. N.; Devine, D.V.; Brooks, D. E. Biomaterials 2008, 29, 1693-1704; Mugabe, C.;Hadaschik, B. A.; Kainthan, R. K.; Brooks, D. E.; So, A. L; Cleave, M.F.; Burt, H. M. BJU Int. 2009, 103, 978-986; Kainthan, R. K.; Brooks, D.E. Bioconjugate Chem. 2008, 19, 2231-2238).

Titration of COOH Groups

Potentiometric/pH titrations, to quantify the total concentration ofHPGs surface-grafted with COOH, were performed on a T-50 M titrator(Mettler Toledo, Mississauga, ON). HPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ andHPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ samples were dissolved at 0.2 mg/ml, in10 mL of 10 mM NaOH. The pH of each solution was manually increased upto approximately 11 by the addition of 0.1 M NaOH. Samples were thentitrated with 0.01 M HCl. Injections were set up in a dynamic range of10-50 μL and a time interval of 30-60 s between injections was to ensureequilibration was established. Titrations were terminated once the pHreached 3.0. Titration end points were determined using the standardextrapolation/intersection method. The reported COOH titration valuesrepresent the mean of three measurements.

The mole ratios of COOH groups conjugated to the HPGs were measured bypotentiometric/pH titration (Table 13) and showed good agreement withtarget mole ratios and the measured molecular weights. For instance, themolecular weight of HPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ can also becalculated by the addition of the number average molecular weight of theHPG-C_(8/10)-MePEG_(6.5) (6.3×10⁴) with the molecular weight ascribed toCOOH groups, which equals the number of carboxylate per HPG molecule (87from titration data) multiplied by the carboxylate molecular weight (101g/mol). Based on this calculation, the number average molecular weightof HPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ is 7.2×10⁴ g/mol, in good agreementwith the measured value (7.0×10⁴ g/mol).

Solubility

The solubility characteristics of HPG polymers were assessed bydissolving known weights of the polymer in various aqueous buffers ordistilled water. The samples were gently vortexed to speed dissolution.The absorbance of polymer solutions at 550 nm was regularly measured forsigns of turbidity for several days to assess whether the polymerremained in solution. For some of the carboxylic acid-derivatized MPGpolymers, the pH of the solution was adjusted to facilitate dissolution.

As potential drug nanocarriers, the solubility characteristics inaqueous media of these polymers are critical. It was found that theHPG-C_(8/10)-MePEG polymer had good water solubility (greater than 100mg/mL) in distilled water, PBS buffer (pH of 7.4), and synthetic urine.HPG-C_(8/10)-COOH was found to be practically insoluble in aqueous mediaor PBS (pH 7.4) and only soluble in alkaline solutions such as 0.1 MNaOH, due to decreased ionization at neutral pH. The hydrophobic (alkylchains) components of the HPG core likely dominated the solubilitycharacteristics. Carboxylate-derivatized HPGs also conjugated with MePEGgroups showed increased water solubility. Accordingly, it was found thatMPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ could be completely dissolved in 10 mMPBS at a concentration of 100 mg/mL without heating, although the pH ofthe solution dropped from 7.4 to 4.5. HPG with a higher amount ofcarboxylate (HPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈) was poorly soluble inwater or PBS buffer and exceeded the buffering capacity, resulting inacidification of PBS buffer and dropping the pH from 7.4 toapproximately 3.8. The solution exhibited significant turbidity asmeasured by absorbance at 550 nm, demonstrating an insoluble residualfraction of polymer (data not shown). The addition of sodium hydroxidewas required to achieve a concentration of 100 mg/mL and a clearsolution at pH 4.25.

Particle Size and Zeta Potential

Particle size and zeta-potential analysis were conducted using a MalvernNanoZS Particle Size analyzer (Malvern Instruments Ltd., Malvern, U.K.)using disposable sizing cuvettes. Polymer solutions at a concentrationof 15 mg/mL were prepared in 1 mM NaCl at pH 6.0 and filtered with a0.22 μm syringe filter (Pall Life Sciences, Ann Arbor, Mich.) prior tomeasurement.

Carboxyl-terminated HPG polymers had particle sizes in the 5-10 nm range(Table 13). Zeta potentials of the nanoparticles were strongly negativeat −41.2±3.2 and −60.3±2.1 mV for HPG-C_(5/10)-MePEG_(6.5)-COOH₁₁₃ andHPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈, respectively. The decrease in zetapotential is attributed to the number of carboxyl groups conjugated tothe surface of the HPGs.

Example 25: Cisplatin Binding to HPGs

The binding of cisplatin to carboxylate modified HPGs was assessed bypreparing 10 mg/mL solutions of the polymers in 0.01 M NaOH. To thesesolutions, cisplatin was added so that the final concentration of drugranged from 0.5 to 4 mg/mL. The pH of each solution was adjusted to 6.0with small volumes of 5 M NaOH. The solutions were incubated overnightat 37° C. with shaking at 50 rpm. Solutions were transferred to Nanosep3K Omega centrifugal filtration devices (Pall Life Sciences, Ann Arbor,Mich.) and centrifuged at 5000 rpm for 10 min. A small volume of thefiltrate (10-40 μL) was diluted to 400 μL with 0.01 M NaOH, and theconcentration of unbound cisplatin in the filtrate was assayed by apreviously described o-phenylenediamine (OPDA) colorimetric assay(Haxton, K. J.; Burt, H. M. Dalton Trans. 2008, 5872-5875). Theconcentration of cisplatin bound to the HPG was determined bysubtracting the concentration of unbound cisplatin found in the filtratefrom the initial concentration of drug added to the HPG.

Binding of cisplatin to the HPGs was achieved through coordination ofthe drug to terminal carboxylate groups on the polymer (FIG. 36). ForHPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ cisplatin bound to the polymer withnearly 100% efficiency up to a maximum of 1 mg/mL (10% w/w; FIG. 37).Above this concentration, free drug was detected in the filtrate,indicating saturation of the carboxylate binding sites and the presenceof unbound drug in the media. HPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ bound upto 2 mg/mL with 100% efficiency before free drug was detected in thefiltrate. This increase in bound drug is attributed to the increasednumber of carboxylate groups and, thus, number of cisplatin bindingsites on HPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ as compared toHPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃.

Example 26: In Vitro Cisplatin Release

Cisplatin was bound to the carboxylate modified HPGs as described abovewith final polymer and cisplatin concentrations of 10 and 1 mg/mL,respectively. Into 7000 MWCO Slide-A-Lyzer mini dialysis units (ThermoScientific, Rockford, Ill.), 20 μL of cisplatin bound polymer solution,or a 1 mg/mL solution of free cisplatin, were added and the samples weredialyzed at 37° C. with stirring against 4 L of 1 mM PBS adjusted to pHsof 4.5, 6.0, and 7.4 or synthetic urine at pH 7.0. Synthetic urine(Surine) was purchased from Dyna-Tek Industries (Lenexa, Kans.). Atpredetermined time points, three dialysis units were removed from therelease media, and the entire contents were removed with three washingsof the dialysis unit followed by dilution to 1 mL with fresh releasemedia. The cisplatin concentration of contents of the dialysis units wasdetermined by OPDA calorimetric assay. The cumulative percent drugreleased was calculated by subtracting the amount of drug remaining fromthe initial amount of drug in the dialysis bag at the beginning of theexperiment. The data were expressed as cumulative percentage of drugreleased as a function of time.

Release of free cisplatin in PBS was rapid and 100% complete within 7 h,demonstrating that the membrane did not impede the release of free drugto any great extent (FIG. 38). For all cisplatin-bound HPG samples, thedrug was thund to release in a controlled fashion, considerably slowerthan the free drug. In PBS, regardless of the pH, cisplatin bound toHPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ was released at nearly the same rate inPBS with approximately 5% released in the first 2 h, 40% release after 1day, and up to 90% released after 7 days. Cisplatin bound toHPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ at pHs of 6.0 and 7.4 released the drugin PBS at similar rates, in a nearly linear manner, with approximately3% of bound cisplatin released in 2 h, 20% released in 1 day, and up to70% over 7 days. The release rate for cisplatin bound toHPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈ at pH 4.5 was faster than its higher pHcounterparts, with a release profile similar to those ofHPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃. The release rate of cisplatin wasconsiderably faster in the presence of urine, with just over 10% of thedose released in 2 h and complete drug release by 2 days forHPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃ and 3 days forHPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈. Similar to release in PBS, thedifference in cisplatin release between the two HPGs may be attributedto the increased number of carboxylate groups present onHPG-C_(8/10)-MePEG_(6.5)-COOH₃₄₈. As urine is a complex mixture made upof several components, it is uncertain which compounds are responsiblefor the increased release of the cisplatin from the HPGs; however, thisincreased release rate may be advantageous, providing a mechanism bywhich the drug release increases upon dilution with urine. Upondisplacement of cisplatin from the HPG it is possible that nitrogencontaining compounds in urine, such as urea, uric acid and creatinine,may bind and inactivate cisplatin. Although cisplatin has been shown tocomplex with these compounds to some degree, it has been determined thatthe majority of cisplatin present in urine after IV administration is inthe originally administered form and the highly active monoaquahydrolysis product (Tang, X.; Hayes Ii, J. W.; Schroder, L.; Cacini, W.;Dorsey, J.; Elder, R. C.; Teppennan, K. Met. Based Drugs 1997, 4,97-109). In light of this finding, it is likely that the majority of thecisplatin released in from the HPGs in urine is in a pharmacologicallyactive form.

Example 27: Cytotoxicity Evaluation

Cytotoxicity studies were performed using the MTS cell proliferationassay (Promega, Madison, Wis.). This assay does not measure immediatecytolytic effects of agents but measures the effect of the polymer oncellular proliferation over long time periods. In this study, KU-7-lucbladder cancer cells, kindly provided by Dr. M. Tachibana (KeioUniversity, Tokyo, Japan). The cells were plated at 5000 cells/well into96-well plates in 180 μL of Dulbecco's modified Eagle (DMEM) medium(Invitrogen Canada, Inc., Burlington, ON) supplemented with 10% fetalbovine serum (FBS) (Invitrogen Canada, Inc., Burlington, ON). 1%penicillin-streptomycin, and 1% L-glutamine and allowed to grow for 24 hat 37° C. in 5% CO₂ to reach approximately 80% confluence forcytotoxicity assays. Cells were then incubated for 2 or 72 h with HPGsalone, ranging from 0.01-100 mg/mL, or free cisplatin orcisplatin-loaded HPGs with drug concentrations ranging from 0.01-100μg/mL. After treatment, the cells were washed twice with Hank's balancedsalt solution (HBSS) and 180 μL of fresh culture media was added intoeach well and cells were allowed to grow for 72 h. Proliferation ofthese cells was measured using a CellTiter 96 aqueous non-radioactivecell proliferation assay (Promega, Madison, Wis.) as describedpreviously (Mugabe, C.; Hadaschik, B. A.; Kainthan, R. K.; Brooks, D.E.; So, A. I.; Gleave, M. E.; Burt, H. M. BJU Int. 2009, 103, 978-986).Briefly, 180 μL of a 10% v/v solution of3-(4,5-dimethythiazol-2-yl)-5-(3-carboxylmethonyphenol)-2-(4-sulfophenyl)-2H-tetrazoliumin HBSS was added to each well and the cells were incubated for 2 h. Theabsorbance was measured at 490 nm with a reference of 620 nm using amicroplate reader.

The inhibition effects of nondrug-loaded HPGs and cisplatin-loaded HPGson KU-7-luc bladder cancer cells were investigated for incubation timesof 2 and 72 h (FIG. 39). These incubation times were chosen to allow forimitation of the typical intravesical instillation period as well as tocompare against previously determined inhibitory concentrations forcisplatin. Inhibitory concentrations at 50% (IC₅₀) for 2 h incubationswere determined to be 1.3, 45.7, 47.0, and 63.0 mg/mL forHPG-C_(8/10)-OH, HPG-C_(8/10)-MePEG_(6.5),HPGC_(8/10)-MePEG_(6.5)-COOH₃₄₈, and HPG-C_(8/10)-MePEG_(6.5)-COOH₁₁₃,respectively. When a 72 h incubation was used, the polymers inhibitedcell proliferation to a greater degree than those found with a 2 hincubation. The HPG-C_(8/10)-OH and HPG-C_(8/10)-MePEG_(6.5) had IC₅₀sof 0.1 and 0.2 mg/mL, respectively. The IC₅₀ for thecarboxylate-modified HPGs decreased approximately 10-fold; however,these polymers still exhibited a high degree of cellular compatibilitywith IC₅₀ values of approximately 5 mg/mL. The overall excellentbiocompatibility of the HPG-C_(8/10)-MePEG and HPG-C_(8/10)-MePEG-COOHprobably arises from the known cellular compatibility of MePEG surfacesensuring little interaction with the plasma membrane of the cells. Theadded benefit of carboxylation may arise from the net negative charge ofthis moiety at a pH of 7.4, establishing a slight repulsive force withthe negatively charged cell surface. Following a 72 h incubation, freecisplatin inhibited KU-7-luc cell proliferation with an IC₅₀ of 1 μg/mL(FIG. 40A), consistent with previous reports for this drug and cellcombination (Hadaschik, B A.; ter Borg, M. G.; Jackson, J.; Sowery, R.D.; So, A. I.; Burt, H. M.; Gleave, M. E. BJU Int. 2008, 101,1347-4355). With a 2 h incubation, this IC₅₀ value increased toapproximately 10 μg/mL (FIG. 40B). When bound toHPG-C_(8/10)-MePEG_(6.5)-COOH polymers, the complexed form of cisplatinalso inhibited KU-7-luc proliferation with higher IC₅₀ values observedfor the 2 h incubation (approximately 50 μg/mL) as compared to the 72incubation values (approximately 5 μg/mL). Clearly, for both 2 and 72 hincubations, the complexed form of cisplatin inhibited cellproliferation less than the free drug by a factor of almost 5. Thisincrease in the IC₅₀ for the drug complexed to the HPGs is likely due tothe slow release rate of the drug from the polymer.

Example 28: Penetration of DTX and Mitomycin F from DifferentFormulations into Pig Bladder Tissue with or without Pre-Treatment

Penetration of DTX from DTX formulations and penetration of mitomycin Ffrom mitomycin F formulations into porcine bladder tissue wereevaluated. Freshly excised porcine bladder tissue sections were mountedon Franz diffusion cells and treated with anticancer drug DTX formulatedin Tween 80, HPG-C_(8/10)-MePEG, or HPG-C_(8/10)-MePEG-NH₂ for 2 hours.In some experiments, the porcine bladder tissue was pretreated withchitosan solution (without drug) or HPG-C_(8/10)-MePEG-NH₂ solution(without drug) for 1 hour before being treated with DTX formulated inTween 80, HPG-C_(8/10)-MePEG or HPG-C_(8/10)-MePEG-NH₂. For mitomycin Fpenetration studies, freshly excised porcine bladder tissue sectionswere mounted on Franz diffusion cells and treated with anticancer drugmitomycin F formulations for 2 hours. The porcine bladder tissue waspretreated with HPG-C_(8/10)-MePEG-NH₂ solution (without drug) for 1hour before being treated with mitomycin F formulations. Tissueconcentration versus tissue depth profiles were obtained and drugexposures were obtained from area-under-the-curve (AUC) calculations.

HPLC-grade acetonitrile and dichloromethane were obtained from FisherScientific (Fairlawn, N.J.). Liquid scintillation fluid, CytoScint™ ES,was purchased from MP Biomedicals (Irvine, Calif.). Tyrode salts werepurchased from Sigma-Aldrich (St. Louis, Mo.) (Tyrodes contains thefollowing in g/L: NaCl: 8.0, KCl: 0.3, NaH₂PO4.5H₂O: 0.093, KH₂PO₄:0.025, NaHCO₃: 1.0, Glucose: 2.0). Docetaxel was obtained from NaturalPharma (Langley BC. Canada). Commercial Taxotere® 20 mg/0.5 mL (SanofiAventis, Laval, QC) was purchased from the BC Cancer Agency at theVancouver General Hospital. Tritium labeled DTX in ethanol was purchasedfrom Moravek Biochemicals (Brea, Calif.) with a specific activity of23.2 Ci/mmol. HPG-C_(8/10)-MePEG was prepared by adapting the protocoldescribed in Example 10 and HPG-C_(8/10)-MePEG-NH₂ was prepared byadapting the protocol described in Example 18. Chitosan was supplied byNovamatrix FMC. Porcine bladders were purchased from Britco Inc.(Langley, BC). Freshly excised urinary bladders were removed on-sitefrom 6-10 month old male pigs weighing between 90-113 kg.

The mots of amine per mol of HPG-MePEG-NH₂ was measured for theHPG-MePEG-NH₂ polymers using different methods, including a forwardtitration method, a back titration method and a fluorescamine assay(Table 14).

TABLE 14 Mol of amine per mol of HPG-MePEG-NH₂ measurements Forwardtitration Fluorescamine method^(a) Back titration method^(b)derivatization method^(c) Sample (mol amine/mol HPG) (mol amine/mol HPG)mol amine/mol HPG) HPG-MePEG-NH₂ 6.7 10.3 10.3 (low) HPG-MePEG-NH₂ 25.2Not done 37.6 (high) ^(a)Forward titration method-titrated against HCl^(b)Back titration method-titrated against NaOH after addition of aknown amount of HCl ^(c)Fluorescence quantitation after derivatizationwith fluorescamine (fluorescamine assay described in Example 15)Preparation of DTX Loaded HPG-C_(8/10)-MePEG, HPG-C_(8/10)-MePEG-NH₂ andTween 80 Formulations

HPG-C_(8/10)-MePEG and HPG-C_(8/10)-MePEG-NH₂ loaded with DTX wereprepared using the solvent evaporation technique. DTX andHPG-C_(8/10)-MePEG or HPG-C_(8/10)-MePEG-NH₂ were dissolved inacetonitrile and dried in an oven at 60° C. for 1 h and flashed withnitrogen to eliminate traces of the organic solvent. Prior to drying,the polymer/drug solution was spiked with a small aliquot of ³H DTX. Theresulting polymer/drug matrix was reconstituted with 60° C. tyrodebuffer (pH 7.4) and vortexed for 2 min. The final concentration of drugwas 0.5 mg/mL and was used at 37° C. DTX was prepared in Tween 80 bydiluting Taxotere® concentrated solution (containing 40 mg of DTX and1040 mg of Tween 80 per mL) with tyrode buffer to yield a finalconcentration of 0.5 mg/mL DTX. Solutions were doped with a small amountof ³H DTX prior to dilution.

Preparation of Mitomycin F Formulations

The mitomycin F (MW=363.4) was prepared in Tyrode's buffer. It wasreceived from American Radiolabeled Chemicals Inc (St Louis, Mo.) Cat#ART-1689. The activity was 1-10 Ci/mmol, 1 mCi/mL in ethanol. Thesolution was prepared by dissolving 50 uL of the ethanolic stock into 3mL of buffer, a 300× dilution.

Preparation of Chitosan Solution and HPG-C_(8/10)-MePEG-NH₂ Solution foruse as a Pretreatment

Chitosan used to prepare the chitosan solutions is PROTASAN™ UP CL 213(Product#: 4210106) and is based on a chitosan where between 75-90% ofthe acetyl groups are removed. The cationic polymer is a highly purifiedand well-characterized water-soluble chloride salt. Typically, themolecular weight for PROTASAN™ UP CL 213 is in the 150000-400000 g/molrange (measured as a chitosan acetate). The chitosan solution wasprepared by dissolving it in water to a solution concentration of 0.5%w/v.

The HPG-C_(8/10)-MePEG-NH₂ solution for use as a pretreatment wasprepared by dissolving HPG-C_(8/10)-MePEG-NH₂ in acetonitrile. Theresulting solution was dried in an oven at 60° C. for 1 h and flashedwith nitrogen to eliminate traces of the organic solvent. The resultingpolymer was reconstituted with 60° C. tyrode buffer (pH 7.4) andvortexed for 2 min.

Tissue Preparation

Freshly excised porcine bladders were removed of excess adipose tissueon the exterior wall and opened longitudinally into left and rightlateral sides and cut into pieces approximately 2 cm×2 cm in a shallowbath of 37° C. tyrode buffer bubbled with carbogen (95% O₂/5% CO₂). Allstudies were performed within 5 h after sacrifice. Bladder pieces weremounted onto a Franz diffusion cell apparatus, such that the luminalside of the bladder wall was exposed to the drug solution. These tissuesections were not stretched and measured approximately 2-3 mm thick.Receptor chambers were filled with 10 mL of 37° C. tyrode buffer (pH7.4). Excess tissue was trimmed around the perimeter of the diffusioncell. The donor chamber of the diffusion cell was filled with 1 mL of0.5 mg/ml drug solution and the tissue exposure area was 0.64 cm². Eachdiffusion cell was set into a shallow water bath and incubated at 37° C.for 2 hours. For some experiments, tissues samples were pre-treated witha chitosan solution (without drug) or a HPG-C_(8/10)-MePEG-NH₂ solution(without drug) for 1 hour before being treated with the DTX or mitomycinF loaded formulations. Tissue samples were washed three times withtyrode buffer to remove all unbound drug. Tissue samples were trimmedand rapidly frozen on metal plates with liquid nitrogen on a bed of dryice.

Cryotome Sectioning of Tissue

Frozen bladder tissue was mounted with Shandon Cryomatrix™ (ThermoScientific, Pittsburgh, Pa.) onto a cryotome object holder. Bladdertissue was sectioned with Shandon MB35 Premier Low Grade MicrotomeBlades (Thermo Scientific, Pittsburgh, Pa.) at −20° C. on a ShandonCryotome Electronic (Thermo Electron Corporation, Cheshire, England)with a R404A refrigeration system. Tissues were sectioned. Tissuesections were placed in pre-weighed 1.5 mL eppendorf tubes and storedfrozen at −20° C.

Quantification of Drug in Tissue

Two hundred μL of acetonitrile was added to the weighed tissue slicesfor drug extraction. Samples were vortexed until all tissue slices werefreely submerged in acetonitrile and left at room temperature for 24hours to ensure complete extraction of drug. The extracted samplesincluding all tissue slices were transferred to scintillation vials and5 mL of scintillation fluid was added. Counts of ³H DTX or ³H mitomycinF were measured by liquid scintillation counting and quantitated usingcalibration graphs from the original stock solution.

Analysis of Tissue Level-Depth Profiles

The tissue level-depth profiles were analyzed for average DTX ormitomycin F concentrations in all layers of the bladder wall down to themuscle, for example, urothelium, lamina propria, and muscularis. Theaverage tissue levels were determined as the total amount of drug foundin the tissue layer divided by the total tissue weight for that layer.The area under the tissue-level depth profile (AUC) was calculated usingthe linear trapezoid rule, as follows:

${AUC}_{0}^{t} = {\sum\limits_{i = 0}^{n - 1}\;{\frac{\left( {t_{i + 1} - t_{i}} \right)}{2} \times \left( {C_{1} + C_{i + 1}} \right)}}$Where, t is tissue depth in μm and C is concentration in μg/gAn estimation by extrapolation of the drug concentration (μg/g) at 0 μmwas required in order to calculate the AUC.

Data for DTX penetration into porcine bladder tissue from DTXformulations without pretreatment are shown in FIG. 41. Both dHPGsevaluated that contained amine resulted in higher drug concentration todepths of about 1500 μm, compared with formulations without amine,including the Tween 80 formulation (Taxotere®) and a dHPG that containedno amine functionality. The effect of adding amine was observed to beconcentration dependent. FIG. 41 shows that the highest concentration ofdrug in tissue, in particular at depths to about 1500 μm was obtainedusing the dHPG formulation that contained the highest amine content (37mol/mol). AUCs at the indicated tissue depth ranges have been calculated(Tables 15a and 15b), showing improvement of dHPG formulations overTaxotere in the range of 1.3 to 2.4 fold. Cavg and Cmax values at theindicated tissue depth ranges have been calculated (Table 15c).

TABLE 15a AUC(180-2640 um) calculated for penetration of docetaxel withdelivery in various vehicles without pretreatment Tween HPG-MePEGHPG-MePEG-NH2 HPG-MePEG-NH2 80 (0 mol) (10 mol) (37 mol) AUC(180-2640)46642 26589 60207 100353 Fold Change of Taxotere 1 0.6 1.3 2.2 SD 5735N/A 30088 21652 N of Runs 4 1 2 2

TABLE 15b AUC(180-1560 um) calculated for penetration of docetaxel withdelivery in various vehicles without pretreatment HPG- HPG- TweenHPG-MePEG MePEG-NH2 MePEG-NH2 80 (0 mol) (10 mol) (37 mol) AUC(180-1560)35874 21983 43768 86899 Fold Change 1 0.6 1.2 2.4 SD 3053 N/A 2054223196 N of Runs 4 1 2 2

TABLE 15c Cavg and Cmax values calculated for the ranges of 180-2640 and180-1560 tissue depth (um), for penetration of docetaxel with deliveryin various vehicles without pretreatment Tween HPG-MePEG HPG-MePEG-NH2HPG-MePEG-NH2 80 (0 mol) (10 mol) (37 mol) C max/avg values (180- 2640)C(max) 35.2 105.6 71.5 22.2 C(avg) 20.3 56.1 33.3 11.8 Fold Change 1.02.8 1.6 0.6 St Dev. C(avg) 8.5 38.1 13.2 6.1 C max/avg value (180-1560)C(max) 35.2 105.6 71.5 22.2 C(avg) 25.4 79.6 39.4 15.5 Fold Change 1.03.1 1.5 0.6 St Dev. C(avg) 5.3 23.6 12.6 3.3

Data for DTX penetration into porcine bladder tissue from DTXformulations with chitosan or HPG-C_(8/10)-MePEG-NH₂ pretreatment areshown in FIGS. 42 and 43. The goal of the experiment was todifferentiate the dHPG polymer's function from chitosan, which is also apolymer containing amine functions. Chitosan has been contemplated as apolymer for use in intravesical delivery; however in the context of apre-treatment to facilitate drug uptake into tissues, it is demonstratedthat dHPGs of certain composition are superior. The data show that withchitosan pre-treatment, the Tween 80 formulation (Taxotere®) providesrelatively little tissue penetration, providing the lowest tissueconcentration for docetaxel in bladder at all tissue depths. Similarly,pre-treatment with chitosan resulted in a modest improvement in tissuepenetration of docetaxel when the drug was administered in theHPG-C_(8/10)-MePEG (no amine) vehicle, however, this was only superiorto the Taxotere-treated (chitosan pre-treated) group. A superior effectin docetaxel tissue penetration was observed when dHPGs containingamines (37 mol amine/mol polymer) (called HPG-C_(8/10)-MePEG-NH₂ inFIGS. 42 and 43). As well, chitosan provided no benefit to delivery whenit was used as a pre-treatment for the docetaxel loadedHPG-C_(8/10)-MePEG-NH2 formulation. The performance of each was comparedby measuring the area under the curve (AUC, FIG. 43) of tissueconcentration over tissue depth (FIG. 42), which is a measure of totaldrug exposure. The results show that the greatest exposure was obtainedwhen pre-treatment or treatment utilized the dHPG containing amine. AUCvalues calculated for the range of 180-3360 tissue depth (um), and Cavgand Cmax values calculated for the ranges of 180-1560 and 180-3360tissue depth (um), for penetration of docetaxel from variousformulations after various pre-treatment regimens are shown in Tables15d and 15e.

TABLE 15d AUC(180-3360 um) calculated for penetration of docetaxel withdelivery in various vehicles after various pre-treatment regimensTaxotere-No Pretreatment Tween 80 HPG-MePEG- HPG-MePEG- (average of 4(Taxotere) - HPG-MePEG - Taxotere - HPG- NH2 -No NH2 - Group: runs)Chitosan Chitosan MePEG-NH2 pretreatment Chitosan Pre-Treatment NoneChitosan Chitosan HPG-MePEG- None Chitosan NH2 Delivery TaxotereTaxotere HPG- Taxotere HPG-MePEG- HPG-MePEG- Vehicle MePEG NH2 NH2Number of 4 × 5 5 5 5 5 5 values replicates Mean 46642 62282 113817149441 142930 154003 Fold Increase 0.8 1 1.8 2.4 2.3 2.5 Over “Taxotere-Chitosan” Fold Increase 1 1.3 2.3 3.0 2.9 3.1 Over “Taxotere-NoPretreat” Std. Deviation 24996 34271 44998 19627 23451 Std. Error 1117815327 20124 8778 10488

TABLE 15e Cavg and Cmax values calculated for the ranges of 180-1560 and180-3360 tissue depth (um), for penetration of docetaxel with deliveryin various vehicles after various pre-treatment regimens Taxotere - HPG-HPG- Tween 80 HPG- MePEG- MePEG- HPG- (Taxotere) - MePEG- NH₂ -No NH₂ -MePEG- Chitosan NH₂ pretreatment Chitosan Chitosan Values for DepthRange: 180-1560 um C(avg) Values 38.47 89.08 91.39 93.64 67.85 Foldincrease 1 2.3 2.4 2.4 1.8 over Taxotere- Chitosan Group St. Deviation16.33 30.78 30.58 30.94 23.44 C(max) Values 61 144.8 123.9 139.5 110.3Fold increase 1 2.4 2.0 2.3 1.8 over Taxotere- Chitosan Group Values forDepth Range: 180-3360 um C(avg) Values 22.55 52.76 51.94 55.07 40.18Fold increase 1 2.3 2.3 2.4 1.8 over Taxotere- Chitosan Group St.Deviation 20.79 45.28 47.95 47.53 34.49 C(max) Values 61 144.8 123.9139.5 110.3 Fold increase 1 2.4 2.0 2.3 1.8 over Taxotere- ChitosanGroupData for mitomycin F penetration into porcine bladder tissue withHPG-C_(8/10)-MePEG-NH₂ pretreatment are shown in FIG. 44.SEM Images of Ex Vivo Penetration Studies of Pig Bladders

The effects of drug penetration observed in Example 28 above werecorrelated with the appearance of the bladder tissue after exposure tothe various treatments. For these experiments, no drug was used and onlya single exposure to a single vehicle per bladder was used. After the 2h exposure time, bladder tissue was harvested, rinsed with buffer andfixed overnight with 4% paraformaldehyde and 2% glutaraldehyde,post-fixed with 1% OsO₄, dehydrated in ethanol and critical point dried.Whole bladders were divided in two and sputter coated with gold. Theentire surface inspected by SEM (Hitachi S4700, 3-5 kV) andrepresentative images were recorded (minimum 3 fields per sample).Representative images are shown (approximately 130 μm wide×100 μm tall).

The SEM images reveal that the urothelium of bladders treated with onlybuffer, and also bladders treated with HPG-C_(8/10)-MePEG (no amine)showed intact or largely intact urothelium, eg no loss of umbrellacells. In contrast, after exposure to the chitosan pre-treatmentvehicle, the surface of the bladder was quite different in appearance,with the urothelium having lost its top layer of umbrella cells (FIG.45). After exposure to HPG-C_(8/10)-MePEG-NH₂ having 10 mol amine/molHPG at solution concentrations of 1 and 10% w/v, partial loss ofumbrella cells from the urothelium was observed (FIG. 46). In contrast,when a HPG-C_(8/10)-MePEG-NH₂ with higher amine content (37 mol/mol) wasused more umbrella cell loss could be seen. The effect was observed tobe concentration dependent. A solution concentration of 0.1% w/vresulted in little or no change in appearance of the bladder surface,whereas as partial and complete loss of umbrella cells was observedafter treatment with solutions having concentrations of 1 and 10% w/vrespectively (FIG. 46). Without being bound by theory, it is believedthat the effect of the amine may be to alter the surface of the bladder,effecting a change in its permeability to the drug. The effect was shownto be dependent on amine content and on polymer concentration.

Example 29: In Vivo Penetration Studies of Mouse Bladders

The effect of exposure of different formulations (without drug) on mousebladders was evaluated. Eight-eleven week old female athymic nude mice(Harlan, Indianapolis, Ind.) were anesthetised to a deep plane using 4%isoflurane and 2 L/min O₂. The bladder was fully expressed andformulations were instilled via a surgically implanted catheter. Apolypropylene purse-string suture was placed around the urethral meatusbefore a lubricated 24-gauge Jelco angiocatheter (BDickenson) was passedthrough the urethra into the bladder. A volume of 50 μL was injected,the animals were slightly inverted (cranially) and the purse-stringsuture was tied off while the catheter was removed in one quick motion.Mice remained anesthetised (at 1.5-2% isoflurane 2 L/min O₂) until the 2hour instillation was completed. The purse-string suture was removed,and the animals were allowed to recover.

Dosing solutions prepared were clear colorless to slightly ambersolutions. The polymer solution concentration was either 1 or 10% w/v.HPG-MePEG polymer was used, which had no amine content, and twoHPG-MePEG-NH2 polymers were used with 8-10 (low) and 37 mol (high) ofamine per mole of HPG (based on a nominal HPG molecular weight of 65 kg/mol). The dosing concentration results are summarized in Table 16.

TABLE 16 Formulation concentration summary Polymer Dosing Solutionconcentration Appearance Amine content PBS 0 Clear, colorless NT^(†)HPG-MePEG 10 Clear, colorless NT (0) HPG-MePEG-NH₂ (10) 10 HPG-MePEG-NH₂(37) 1 HPG-MePEG-NH₂ (37) 10 ^(†)NT means not tested.

Bladders were excised from each mouse for SEM and histology. All animalswere observed post-administration for 2 hours and prior to each tissuecollection for mortality and morbidity. No signs of mortality ormorbidity were noted, with the exception of some small amount of bloodat the instillation site.

SEM Analysis

Tissues were washed 3 times with PBS, fixed overnight in 2%paraformaldehyde then transferred in 0.1 M cacodylate buffer. Tissueswere postfixed in 1% osmium tetroxide for 1 h at room temperature thendehydrated in ethanol mixed with water, in increasing percentage ofethanol in the mixture, starting at 30 and increasing to 100%. Sampleswere dried by critical point dehydration and then sputter-coated withgold-palladium twice (once at 90° and second time at 45°). Samples wereexamined in scanning electron microscopy with Hitachi S4700 atBioimaging Facility. Each bladder was visualized at low magnificationand then the entire surface examined again at high magnification.Multiple (9-10 images) were taken at various magnifications

TABLE 17 Formulation concentration summary Amine Density Polymer Hours(mol/mol Concentration post-instillation Formulation HPG) (% w/v) 2 6 24PBS N/A^(†) N/A Intact NT^(††) NT HPG-MePEG 0 10 Intact Intact IntactHPG-MePEG-NH₂ 10 (low) 10 Intact + Intact 37 (high) 1 ++ Intact + 37(high) 10 ++ ++ Intact ^(†)N/A means not applicable. ^(††)NT means nottested. Intact: no signs of umbrella cell loss +: very little or partialloss of umbrella cells ++: substantial or complete loss of umbrellacells

The mouse bladder surface treated with a 2 hour instillation of PBS hadan intact umbrella cell layer and had a folded appearance as seen in theSEM image of the mouse bladder surface in FIG. 47. The mouse bladdersurface treated with a 2 hour instillation of HPG-MePEG solution at 10%w/v had an intact umbrella cell layer immediately after the 2 hourinstillation period. An intact umbrella cell layer was also observed at6 hours and 24 hours after the HPG-MePEG solution instillation (FIG.48). As shown in FIG. 48, the surface cells had a flat appearance. Themouse bladder surface treated with a 2 hour instillation of theHPG-MePEG-NH₂ (10 mol/mol) 10% w/v solution had an intact umbrella celllayer immediately after the 2 hour instillation period (FIG. 49). At 6hours after the HPG-MePEG-NH₂ (10 mol/mol) 10% w/v solutioninstillation, the mouse bladder surface exhibited loss of a singleumbrella cell, exposing lower layers of epithelium. At 24 hours afterthe same instillation, the mouse bladder surface had an intact umbrellasurface layer (FIG. 49). The mouse bladder surface treated with a 2 hourinstillation of the HPG-MePEG-NH₂ (37 mol/mol) 1% w/v solution exhibitedcomplete loss of umbrella cells, exposing lower layers of epitheliumimmediately after the 2 hour instillation period as shown in FIG. 50. At6 hours after the HPG-MePEG-NH₂ (37 mol/mol) 1% w/v solutioninstillation, the mouse bladder surface still exhibited substantial lossof umbrella cells. At 24 hours after the HPG-MePEG-NH₂ (37 mol/mol) 1%w/v solution instillation, the mouse bladder surface had a partiallyintact surface (upper section of FIG. 50C) with significant loss ofumbrella cells (lower left of FIG. 50C). The mouse bladder surfacetreated with the 2 hour instillation of HPG-MePEG-NH₂ (37 mol/mol) 10%solution exhibited a complete loss of umbrella cells, exposing lowerlayers of epithelium immediately after the 2 hour instillation period asshown in FIG. 51. At 6 hours after the HPG-MePEG-NH₂ (37 mol/mol) 10%solution instillation, the mouse bladder surface also exhibited completeloss of umbrella cells. At 24 hours after the instillation, the mousebladder surface had an intact umbrella cell layer; however, the surfacecells appeared smaller and less flat in appearance than other observedintact layers (FIG. 51). The effect of the formulations on umbrella cellloss was observed to be dependent on amine content of the dHPG and ondHPG concentration.

Histology

Tissues were evaluated for changes in tight junctions, exfoliation ofcells, and infiltration of inflammatory cells. Histological analysisresults are summarized in Table 18. As can be seen from the histologyresults, signs of inflammation and necrosis were not observed in themouse bladder surface after exposure to the dHPG formulations.

TABLE 18 Histology of Mice Bladder Surface Amine Solution Sample Signscontent Concentration time Neutrophil Signs of of Formulation (mol/mol)(% w/v) (h) infiltration inflammation necrosis Untreated N/A N/A 2 No NoNo PBS N/A N/A 2 No No No HPG- 0 10 2 No No No MePEG 6 No No No 24 No NoNo HPG- 10 (Low) 10 2 No No No MePEG- 6 No No No NH₂ 24 No No No 24 NoNo No 37 (High)  1 2 No No No 6 No No No 24 No No No 24 No No No 10 6 NoNo No 6 No No No 24 No No NoUrine Analysis

Urine was collected at the time of sacrifice. After euthanizing theanimal, the bladder was exposed and its contents removed by bladderpuncture and withdrawal through a 25 G needle. The urine was stored onice (but not frozen) and transported for evaluation. Urine was analyzedfor the presence of cells. A few drops of urine was placed onto amicroscope slide and observed by microscope for the presence of cells.Any cells present were counted with a hemocytometer slide. Cells countsin urine harvested from mice immediately after the 2 hour instillation,and at the time of bladder harvest (2, 6, 24 hours), are shown in FIG.52.

Blood Analysis

Blood was collected upon termination by CO₂ inhalation by cardiacpuncture upon last breath, approximately 500-700 μL was placed into EDTAmicrotainer tubes. Each tube was inverted several times to ensure evenmixing of blood and EDTA to prevent coagulation. Blood samples werestored on ice until all samples were collected for a particular timepoint and then processed to generate plasma. Plasma was generated bycentrifuging samples at 2500 rpm for 15 minutes at 4° C. (rpm based onBeckman 3.8A rotor, RCF_(avg) 200×g). The plasma supernatant waspipetted off and placed into labelled vials and stored at −80° C. Bloodwas analyzed for TNFα levels using the MesoScale platform and standardassay kits. Circulating TNFα levels in the mouse blood at 2, 6 and 24 hafter instillation with the various formulations are shown in FIG. 53.No TNFα was detected in any of the samples.

Although various embodiments of the invention are disclosed herein, manyadaptations and modifications may be made within the scope of theinvention in accordance with the common general knowledge of thoseskilled in this art. Such modifications include the substitution ofknown equivalents for any aspect of the invention in order to achievethe same result in substantially the same way. Numeric ranges areinclusive of the numbers defining the range. The word “comprising” isused herein as any open-ended term, substantially equivalent to thephrase “including, but not limited to”, and the word “comprises” has acorresponding meaning. As used herein, the singular forms “a”, “an” and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a thing” includes more thanone such thing.

Citation of references herein is not an admission that such referencesare prior art to the present invention nor does it constitute anyadmission as to the contents or date of these documents. Any prioritydocument(s) and all publications, including but not limited to patentsand patent applications, cited in this specification are incorporatedherein by reference as if each individual publication were specificallyand individually indicated to be incorporated by reference herein and asthough fully set forth herein. The invention includes all embodimentsand variations substantially as hereinbefore described and withreference to the examples and drawings.

What is claimed is:
 1. A hyperbranched polyglycerol comprising: a corecomprising hyperbranched polyglycerol derivatized with C₈ alkyl chains,C₁₀ alkyl chains, or a combination thereof, wherein the ratio of alkylchains to glycerol units is greater at a center of the core compared toa periphery of the core; and a shell comprising at least one hydrophilicsubstituent and at least one functional group, wherein the at least onehydrophilic substituent comprises methoxypolyethylene glycol (MePEG),polyethylene glycol (PEG), or a combination thereof, and the at leastone functional group comprises —NH₂ or —NH₃ ⁺.
 2. The hyperbranchedpolyglycerol according to claim 1, wherein the hyperbranchedpolyglycerol comprises from about 1 to about 200 moles of the at leastone functional group per mole of the hyperbranched polyglycerol.
 3. Thehyperbranched polyglycerol according to claim 1, wherein thehyperbranched polyglycerol comprises from about 1 to about 100 moles ofthe at least one functional group per mole of the hyperbranchedpolyglycerol.
 4. The hyperbranched polyglycerol according to claim 1,wherein the hyperbranched polyglycerol comprises from about 1 to about40 moles of the at least one functional group per mole of thehyperbranched polyglycerol.
 5. The hyperbranched polyglycerol accordingto claim 1, wherein the hyperbranched polyglycerol comprises from about5 to about 40 moles of the at least one functional group per mole of thehyperbranched polyglycerol.
 6. The hyperbranched polyglycerol accordingto claim 1, wherein the hyperbranched polyglycerol comprises from about5 to about 15 moles of the at least one functional group per mole of thehyperbranched polyglycerol.
 7. The hyperbranched polyglycerol accordingto claim 1, wherein the hyperbranched polyglycerol comprises from about1 to about 200 moles of the at least one hydrophilic substituent permole of the hyperbranched polyglycerol.
 8. The hyperbranchedpolyglycerol according to claim 1, wherein the hyperbranchedpolyglycerol comprises from about 1 to about 100 moles of the at leastone hydrophilic substituent per mole of the hyperbranched polyglycerol.9. The hyperbranched polyglycerol according to claim 1, wherein thehyperbranched polyglycerol comprises from about 1 to about 40 moles ofthe at least one hydrophilic substituent per mole of the hyperbranchedpolyglycerol.
 10. The hyperbranched polyglycerol according to claim 1,wherein the hyperbranched polyglycerol comprises from about 10 to about40 moles of the at least one hydrophilic substituent per mole of thehyperbranched polyglycerol.
 11. The hyperbranched polyglycerol accordingto claim 1, wherein the hyperbranched polyglycerol comprises from about10 to about 30 moles of the at least one hydrophilic substituent permole of the hyperbranched polyglycerol.
 12. The hyperbranchedpolyglycerol according to claim 1, wherein the core compriseshyperbranched polyglycerol derivatized with C₈ alkyl chains and C₁₀alkyl chains.
 13. The hyperbranched polyglycerol according to claim 1,wherein the core comprises hyperbranched polyglycerol derivatized withC₈ alkyl chains.
 14. The hyperbranched polyglycerol according to claim1, wherein the core comprises hyperbranched polyglycerol derivatizedwith C₁₀ alkyl chains.
 15. The hyperbranched polyglycerol according toclaim 1, wherein the at least one hydrophilic substituent comprisesMePEG and PEG.
 16. The hyperbranched polyglycerol according to claim 1,wherein the at least one hydrophilic substituent comprises PEG.
 17. Thehyperbranched polyglycerol according to claim 1, wherein the at leastone hydrophilic substituent comprises MePEG.
 18. The hyperbranchedpolyglycerol according to claim 1, wherein the at least one functionalgroup comprises —NH₂.
 19. The hyperbranched polyglycerol according toclaim 1, wherein the at least one functional group comprises —NH₃ ⁺. 20.The hyperbranched polyglycerol according to claim 1, further comprisinga biologically active moiety selected from the group consisting ofdocetaxel, paclitaxel, valrubicin, vinblastine, mitomycin, cisplatin,methotrexate, doxorubicin, epirubicin, gemcitabine, everolimus, suramin,and combinations thereof.
 21. A pharmaceutical composition comprising ahyperbranched polyglycerol according to claim 20 and a pharmaceuticallyacceptable carrier.
 22. A method of treating a cancer in a subject inneed thereof, the method comprising administering a hyperbranchedpolyglycerol according to claim 20 to the subject, wherein theadministering is effective to treat a cancer in the subject, wherein thecancer is a bladder cancer.
 23. The method according to claim 22,wherein the cancer is non-muscle invasive bladder cancer.
 24. The methodaccording to claim 22, wherein the administering comprises intravesicaladministration of the hyperbranched polyglycerol.
 25. The methodaccording to claim 22, wherein the subject is a human.
 26. A method ofdelivering a biologically active moiety to a biological tissue of apatient, the method comprising administering a hyperbranchedpolyglycerol according to claim 20 to the patient, wherein theadministering is effective to increase uptake of the biologically activemoiety by the biological tissue of the patient.
 27. A method ofincreasing permeability of a biological tissue of a patient to abiologically active moiety, the method comprising administering ahyperbranched polyglycerol according to claim 20 to the patient, whereinthe administering is effective to increase permeability of thebiological tissue of the patient to the biologically active moiety. 28.A pharmaceutical composition comprising a hyperbranched polyglycerolaccording to claim 1 and a pharmaceutically acceptable carrier.
 29. Apharmaceutical composition comprising a hyperbranched polyglycerolaccording to claim 1 and a biologically active moiety selected from thegroup consisting of docetaxel, paclitaxel, valrubicin, vinblastine,mitomycin, cisplatin, methotrexate, doxorubicin, epirubicin,gemcitabine, everolimus, suramin, and combinations thereof.
 30. Thepharmaceutical composition according to claim 29, further comprising apharmaceutically acceptable carrier.
 31. The pharmaceutical compositionaccording to claim 29, wherein the biologically active moiety isdocetaxel.
 32. The pharmaceutical composition according to claim 29,wherein the biologically active moiety is paclitaxel.
 33. Thepharmaceutical composition according to claim 29, wherein thecombination of moieties is methotrexate, vinblastine, and doxorubicin,or methotrexate, vinblastine, doxorubicin and cisplatin.
 34. Thepharmaceutical composition according to claim 29, wherein thehyperbranched polyglycerol comprises from about 1 to about 200 moles ofthe at least one functional group per mole of the hyperbranchedpolyglycerol.
 35. The pharmaceutical composition according to claim 29,wherein the hyperbranched polyglycerol comprises from about 1 to about40 moles of the at least one functional group per mole of thehyperbranched polyglycerol.
 36. The pharmaceutical composition accordingto claim 29, wherein the hyperbranched polyglycerol comprises from about5 to about 15 moles of the at least one functional group per mole of thehyperbranched polyglycerol.
 37. The pharmaceutical composition accordingto claim 29, wherein the hyperbranched polyglycerol comprises from about1 to about 200 moles of the at least one hydrophilic substituent permole of the hyperbranched polyglycerol.
 38. The pharmaceuticalcomposition according to claim 29, wherein the hyperbranchedpolyglycerol comprises from about 1 to about 40 moles of the at leastone hydrophilic substituent per mole of the hyperbranched polyglycerol.39. The pharmaceutical composition according to claim 29, wherein thehyperbranched polyglycerol comprises from about 10 to about 30 moles ofthe at least one hydrophilic substituent per mole of the hyperbranchedpolyglycerol.
 40. The pharmaceutical composition according to claim 29,wherein the core comprises hyperbranched polyglycerol derivatized withC₈ alkyl chains and C₁₀ alkyl chains.
 41. The pharmaceutical compositionaccording to claim 29, wherein the core comprises hyperbranchedpolyglycerol derivatized with C₈ alkyl chains.
 42. The pharmaceuticalcomposition according to claim 29, wherein the core compriseshyperbranched polyglycerol derivatized with C₁₀ alkyl chains.
 43. Thepharmaceutical composition according to claim 29, wherein the at leastone hydrophilic substituent comprises MePEG and PEG.
 44. Thepharmaceutical composition according to claim 29, wherein the at leastone hydrophilic substituent comprises PEG.
 45. The pharmaceuticalcomposition according to claim 29, wherein the at least one hydrophilicsubstituent comprises MePEG.
 46. The pharmaceutical compositionaccording to claim 29, wherein the at least one functional groupcomprises —NH₂.
 47. The pharmaceutical composition according to claim29, wherein the at least one functional group comprises —NH₃ ⁺.
 48. Amethod of treating a cancer in a subject in need thereof, the methodcomprising administering a pharmaceutical composition according to claim29 to the subject, wherein the administering is effective to treat acancer in the subject, wherein the cancer is a bladder cancer.
 49. Themethod according to claim 48, wherein the cancer is non-muscle invasivebladder cancer.
 50. The method according to claim 48, wherein theadministering comprises intravesical administration of thepharmaceutical composition.
 51. The method according to claim 48,wherein the subject is a human.
 52. The method according to claim 48,wherein: (a) the at least one functional group is present in an amountof from about 1 to about 40 moles of the at least one functional groupper mole of the hyperbranched polyglycerol and the at least onehydrophilic substituent is present in an amount of from about 1 to about40 moles of the at least one hydrophilic substituent per mole of thehyperbranched polyglycerol; or (b) the at least one functional group ispresent in an amount of from about 5 to about 15 moles of the at leastone functional group per mole of the hyperbranched polyglycerol and theat least one hydrophilic substituent is present in an amount of fromabout 10 to about 30 moles of the at least one hydrophilic substituentper mole of the hyperbranched polyglycerol.
 53. The method according toclaim 52, wherein the core comprises hyperbranched polyglycerolderivatized with C₁₀ alkyl chains, and the at least one hydrophilicsubstituent comprises MePEG.
 54. The method according to claim 53,wherein the biologically active moiety is docetaxel.
 55. The methodaccording to claim 53, wherein the cancer is non-muscle invasive bladdercancer.
 56. The method according to claim 54, wherein the cancer isnon-muscle invasive bladder cancer.
 57. The method according to claim52, wherein the biologically active moiety is docetaxel.
 58. The methodaccording to claim 52, wherein the cancer is non-muscle invasive bladdercancer.
 59. The method according to claim 48, wherein the core compriseshyperbranched polyglycerol derivatized with C₁₀ alkyl chains, and the atleast one hydrophilic substituent comprises MePEG.
 60. The methodaccording to claim 59, wherein the biologically active moiety isdocetaxel.
 61. The method according to claim 59, wherein the cancer isnon-muscle invasive bladder cancer.
 62. The method according to claim48, wherein the biologically active moiety is docetaxel.
 63. The methodaccording to claim 62, wherein the cancer is non-muscle invasive bladdercancer.
 64. The pharmaceutical composition according to claim 29,wherein: (a) the at least one functional group is present in an amountof from about 1 to about 40 moles of the at least one functional groupper mole of the hyperbranched polyglycerol and the at least onehydrophilic substituent is present in an amount of from about 1 to about40 moles of the at least one hydrophilic substituent per mole of thehyperbranched polyglycerol; or (b) the at least one functional group ispresent in an amount of from about 5 to about 15 moles of the at leastone functional group per mole of the hyperbranched polyglycerol and theat least one hydrophilic substituent is present in an amount of fromabout 10 to about 30 moles of the at least one hydrophilic substituentper mole of the hyperbranched polyglycerol.
 65. The pharmaceuticalcomposition according to claim 64, wherein the core compriseshyperbranched polyglycerol derivatized with C₁₀ alkyl chains, and the atleast one hydrophilic substituent comprises MePEG.
 66. Thepharmaceutical composition according to claim 64, wherein thebiologically active moiety is docetaxel.
 67. The pharmaceuticalcomposition according to claim 65, wherein the biologically activemoiety is docetaxel.
 68. The pharmaceutical composition according toclaim 29, wherein the core comprises hyperbranched polyglycerolderivatized with C₁₀ alkyl chains, and the at least one hydrophilicsubstituent comprises MePEG.
 69. The pharmaceutical compositionaccording to claim 68, wherein the biologically active moiety isdocetaxel.
 70. A method of delivering a biologically active moiety to abiological tissue of a patient, the method comprising administering tothe patient a hyperbranched polyglycerol according to claim 1 and abiologically active moiety selected from the group consisting ofdocetaxel, paclitaxel, valrubicin, vinblastine, mitomycin, cisplatin,methotrexate, doxorubicin, epirubicin, gemcitabine, everolimus, suramin,and combinations thereof, wherein the administering is effective toincrease uptake of the biologically active moiety by the biologicaltissue of the patient.
 71. The method according to claim 70, wherein theadministering comprises pre-treating the biological tissue byadministering the hyperbranched polyglycerol to the patient beforeadministering the biologically active moiety to the patient.
 72. Themethod according to claim 70, wherein the administering comprisesco-treating the biological tissue by administering the hyperbranchedpolyglycerol to the patient during administration of the biologicallyactive moiety to the patient.
 73. The method according to claim 70,wherein: (a) the at least one functional group is present in an amountof from about 1 to about 40 moles of the at least one functional groupper mole of the hyperbranched polyglycerol and the at least onehydrophilic substituent is present in an amount of from about 1 to about40 moles of the at least one hydrophilic substituent per mole of thehyperbranched polyglycerol; or (b) the at least one functional group ispresent in an amount of from about 5 to about 15 moles of the at leastone functional group per mole of the hyperbranched polyglycerol and theat least one hydrophilic substituent is present in an amount of fromabout 10 to about 30 moles of the at least one hydrophilic substituentper mole of the hyperbranched polyglycerol.
 74. The method according toclaim 73, wherein the core comprises hyperbranched polyglycerolderivatized with C₁₀ alkyl chains, and the at least one hydrophilicsubstituent comprises MePEG.
 75. The method according to claim 74,wherein the biologically active moiety is docetaxel.
 76. The methodaccording to claim 73, wherein the biologically active moiety isdocetaxel.
 77. The method according to claim 70, wherein the corecomprises hyperbranched polyglycerol derivatized with C₁₀ alkyl chains,and the at least one hydrophilic substituent comprises MePEG.
 78. Themethod according to claim 77, wherein the biologically active moiety isdocetaxel.
 79. The method according to claim 70, wherein thebiologically active moiety is docetaxel.
 80. The hyperbranchedpolyglycerol according to claim 1, wherein: (a) the at least onefunctional group is present in an amount of from about 1 to about 40moles of the at least one functional group per mole of the hyperbranchedpolyglycerol and the at least one hydrophilic substituent is present inan amount of from about 1 to about 40 moles of the at least onehydrophilic substituent per mole of the hyperbranched polyglycerol; or(b) the at least one functional group is present in an amount of fromabout 5 to about 15 moles of the at least one functional group per moleof the hyperbranched polyglycerol and the at least one hydrophilicsubstituent is present in an amount of from about 10 to about 30 molesof the at least one hydrophilic substituent per mole of thehyperbranched polyglycerol.
 81. The hyperbranched polyglycerol accordingto claim 1, wherein the core comprises hyperbranched polyglycerolderivatized with C₁₀ alkyl chains, and the at least one hydrophilicsubstituent comprises MePEG.
 82. The hyperbranched polyglycerolaccording to claim 80, wherein the core comprises hyperbranchedpolyglycerol derivatized with C₁₀ alkyl chains, and the at least onehydrophilic substituent comprises MePEG.